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 hypoxia and glioma dual-specific suicide gene using dexamethasone conjugated polyethylenimine for glioblastoma-specific gene therapy.

Gene therapy has been considered a promising approach for glioblastoma therapy. To avoid side effects and increase the specificity of gene expression, gene expression should be tightly regulated. In this study, glioma and hypoxia dual-specific plasmids (pEpo-NI2-SV-Luc and pEpo-NI2-SV-HSVtk) were developed by combining the erythropoietin (Epo) enhancer and nestin intron 2 (NI2). In the in vitro studies, pEpo-NI2-SV-Luc showed higher gene expression under hypoxia than normoxia in a glioblastoma-specific manner. The MTT and caspase assays demonstrated that pEpo-NI2-SV-HSVtk specifically induced caspase activity and cell death in hypoxic glioblastoma cells. For in vivo evaluation, subcutaneous and intracranial glioblastoma models were established. Dexamethasone-conjugated-polyethylenimine (PEI-Dexa) was used as a gene carrier, since PEI-Dexa efficiently delivers plasmid to glioblastoma cells and also has an antitumor effect due to the effect of dexamethasone. In the in vivo study in the subcutaneous and intracranial glioblastoma models, the tumor size was reduced more effectively in the pEpo-NI2-SV-HSVtk group than in the control and pSV-HSVtk groups. In addition, higher levels of HSVtk gene expression and TUNEL-positive cells were observed in the pEpo-NI2-SV-HSVtk group compared with the control and pSV-HSVtk groups, suggesting that pEpo-NI2-SV-HSVtk increased the therapeutic efficacy in hypoxic glioblastoma. Therefore, pEpo-NI2-SV-HSVtk/PEI-Dexa complex may be useful for glioblastoma-specific gene therapy.

Targeted gene delivery to ischemic myocardium by homing peptide-guided polymeric carrier.

Myocardial ischemia needs an alternative treatment such as gene therapy for the direct protection of cardiomyocytes against necrosis or apoptosis and to prevent the development of myocardial fibrosis and cardiac dysfunction. Despite the utility of gene therapy, its therapeutic use is limited due to inadequate transfection in cardiomyocytes and difficulty in directing to ischemic myocardium. Here, we present a polymeric gene carrier that is capable of targeting ischemic myocardium, resulting in high localization within the ischemic zone of the left ventricle (LV) of an ischemia/reperfusion (I/R) rat model upon systemic administration. Cystamine bisacrylamide-diamino hexane (CD) polymer was modified with the ischemic myocardium-targeted peptide (IMTP) and D-9-arginine (9R) for dual effects of the homing to ischemic myocardium and enhanced transfection efficiency with minimized polymer use. Conjugation of IMTP and 9R to CD led to an increase in transfection under hypoxia and significantly reduced the amount of polymer required for high transfection. Finally, we confirmed targeting of IMTP-CD-9R/DNA polyplex to ischemic myocardium and enhanced gene expression in LV of the I/R rat after tail vein injection. This study provides a clue that gene therapy for the treatment of myocardial ischemia can be achieved by using homing peptide-guided gene delivery systems.

Hybrid nanoparticles with cell membrane and dexamethasone-conjugated polymer for gene delivery into the lungs as therapy for acute lung injury.

Gene therapy has been suggested as a new treatment for acute lung injury (ALI), which is a severe inflammatory disease. Previously, amphiphilic polymeric carriers such as dexamethasone-conjugated polyethylenimine (PEI) (DP) have been used to transport plasmid DNA (pDNA) into the lungs. In the current study, hybrid nanoparticles comprising DP and cell membrane (CM) from LA-4 lung epithelial cells were developed for enhanced delivery of pDNA into the lungs. The CM components of the hybrid nanoparticles may interact with plasma membranes of target cells and facilitate intracellular uptake of pDNA. DP/CM/pDNA nanoparticles had the highest transfection efficiency into LA-4 cells at a weight ratio of 8 : 3 : 1. In vitro transfection assays showed that DP/CM/pDNA nanoparticles improved the cellular uptake and transfection efficiency of pDNA compared with PEI (25 kDa, PEI25k)/pDNA and DP/pDNA nanoparticles. The DP/CM/pDNA nanoparticles were approximately 80 nm in diameter with a zeta potential of +25 mV. To evaluate the therapeutic effects, heme oxygenase-1 pDNA (pHO-1) was administered to ALI animal models by intratracheal instillation. DP/CM/pHO-1 nanoparticles improved gene delivery efficiency compared with PEI25k/pHO-1 and DP/pHO-1 nanoparticles. As a result, inflammation in the lungs was alleviated by DP/CM/pHO-1 nanoparticles more effectively than by other nanoparticles. The results suggest that DP/CM/pDNA hybrid nanoparticles may be useful gene carriers for the treatment of ALI.

Delivery of self-replicating messenger RNA into the brain for the treatment of ischemic stroke.

Ischemic stroke is caused by the occlusion of cerebral arteries. In the ischemic stroke, ischemia-reperfusion injury increases the damage in the brain after reperfusion. In the previous study, heme oxygenase-1 (HO1) mRNA was delivered into the ischemic brain, showing that HO1-mRNA had higher therapeutic effect and less side-effect than HO1-plasmid (pHO1). However, mRNA is degraded faster than plasmid DNA reducing the duration of gene expression. In this study, self-replicating mRNA (Rep-mRNA) was developed using a replicon system from Venezuelan Equine Encephalitis virus to compensate this disadvantage of mRNA delivery. Deoxycholic acid-conjugated polyethylenimine (DA-PEI) was used as a carrier of the mRNAs. The Rep-mRNA/DA-PEI complex had a size of around 90 nm and a zeta-potential of 33 mV. In the in vitro transfection assays, gene expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 14 days, while that by the HO1-mRNA/DA-PEI complex approached basal level at 3 days after transfection. Therapeutic effects of the HO1-Rep-mRNA/DA-PEI complexes were evaluated in the ischemic stroke animal model. The complexes were injected into the brain stereotaxically. HO1 expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 7 days after injection, but the pHO1/DA-PEI or HO1-mRNA/DA-PEI complex showed basal level of HO1-expression at 7 days after injection. Due to higher and longer expression of HO1, the apoptosis level and infarct size were decreased by the HO1-Rep-mRNA/DA-PEI complexes, compared with the pHO1/DA-PEI and HO1-mRNA/DA-PEI complex. These results suggest that HO1-Rep-mRNA/DA-PEI complex may have a potential as a long-lasting therapeutic system for the treatment of ischemic stroke.

Tonsil mesenchymal stem cells-derived extracellular vesicles prevent submandibular gland dysfunction in ovariectomized rats.

Dry mouth that occurs after menopause significantly reduces the quality of life of the elderly. The extracellular vesicles derived from mesenchymal stem cells are being studied for application in various pathological conditions in the field of tissue regenerative medicine. This study is to investigate the therapeutic effect on salivary gland dysfunction occurring after ovariectomy using tonsil mesenchymal stem cells (T-MSCs)-derived extracellular vesicles. The rats were divided into the following groups: sham-operated rats (SHAM), rats that underwent ovariectomy (OVX), and rats that underwent OVX surgery and were simultaneously injected with T-MSC-derived extracellular vesicles (OVX+EV). The rats were sacrificed 6 weeks after ovariectomy. Estradiol levels decreased in the OVX group compared with those in the SHAM group. Extracellular vesicles had no effect on estradiol levels or estrogen receptor ß expression. The evaluation of pro-inflammatory cytokines, TNF-α and IL-6, increased in the OVX group and decreased in the OVX+EV group. The expressions of collagen I and TGFßI increased in the OVX group but decreased in the OVX+EV group. Moreover, to examine submandibular gland function, AQP5 and α-amylase expressions were downregulated in the OVX group, but improved upon exosome injection. In conclusion, T-MSC-derived extracellular vesicles are useful for the prevented submandibular gland dysfunction that occurs after menopause.

Preparation of dexamethasone-based cationic liposome and its application to gene delivery in vitro.

In this study, dexamethasone was conjugated to PAMAM dendrimer (generation 0) and its gene transfection efficiency was investigated. To make a liposomal solution for gene delivery, DOPE was used as a fusogenic helper lipid. In gel retardation assay, PAMAM-dexamethasone conjugate (PAM-Dex)/DOPE liposome/DNA complex was completely retarded at 8:1 N/P (nitrogen/phosphate) ratio. The physicochemical characteristics are studied by measuring the average size distribution and zeta-potential values of the complexes. In vitro transfection assay showed that the PAM-Dex/DOPE liposome/DNA complex displayed higher gene delivery efficiency compared to PAMAM/DNA complex. In addition, PAM-Dex/DOPE liposome showed the lowest toxicity compared to PAMAM, PEI 25 kD and Lipofectamine. These results indicate that PAM-Dex/DOPE liposome has a potential to be used as an efficient gene carrier for gene therapy.

Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma.

Gene therapy has been introduced as an alternative to radiation and chemical therapy for glioblastoma. Biomimetic nanoparticles coated with cell membranes (CM) have advantages such as high biocompatibility and prolong half-life. To apply CM coated nanoparticles to gene delivery, the polyethylenimine (PEI25k)/plasmid DNA (pDNA) complexes were coated with CM from C6 rat glioblastoma cells. With the CM covering, the PEI25k/pDNA complexes formed stable nanoparticles with negative surface charge. The PEI25k/pDNA/CM nanoparticles had high colloidal stability and could be stored for approximately 20 days without aggregation. The transfection efficiency of the PEI25k/pDNA/CM nanoparticles was higher than that of the PEI25k/pDNA complex in serum-containing medium. This suggests that serum does not interfere with transfection efficiency of the nanoparticles. Moreover, the PEI25k/pDNA/CM nanoparticles had lower toxicity than the PEI25k/DNA complex in vitro and in vivo. The PEI25k/pDNA/CM nanoparticles prepared with CMs of different types of cells were transfected into cells. The results showed that the PEI25k/pDNA/CM nanoparticles with the C6 CM had the highest transfection efficiency to C6 cells, suggesting the homotypic targeting effect. The therapeutic effects of the nanoparticles were evaluated in intracranial C6 transplanted glioblastoma animal models. The PEI25k/pDNA/CM nanoparticles were prepared with herpes simplex virus thymidine kinase plasmid (pHSVtk) and injected into the tumor locally. The results showed that the PEI25k/pHSVtk/CM nanoparticles induced higher HSVtk expression compared with the PEI25k/pHSVtk complex. Furthermore, tumor size was reduced more efficiently by the PEI25k/pHSVtk/CM nanoparticles than by the PEI25k/pHSVtk complex. Overall results indicate that PEI25k/pDNA/CM nanoparticles are suitable for pDNA delivery to glioblastoma.

Prevascularized Tracheal Scaffolds Using the Platysma Flap for Enhanced Tracheal Regeneration.

OBJECTIVES: One of the greatest hurdles in tracheal tissue engineering is insufficient vascularization, which leads to delayed mucosal regeneration, inflammation, and restenosis. This study investigated whether a prevascularized segmental tracheal substitute using platysma can enhance tracheal mucosal regeneration. METHODS: Three-dimensional (3D) printed scaffolds with (group M) or without (group S) Matrigel coating were implanted under the feeding vessels of the platysma in New Zealand White rabbits (n = 3) to induce vascularization. After 1 or 2 weeks, tracheal defects were created and vascularized scaffolds with feeders of the platysma were transplanted as rotational flaps. As controls, scaffolds with or without Matrigel coating was transplanted into a tracheal defect without prevascularization. Airway patency and epithelization were examined using a rigid bronchoscope every 2 weeks. Surviving animals were euthanized at 24 weeks, and microcomputed tomography and histological evaluation were performed. RESULTS: Animals with 2 weeks of prevascularization showed longer survival than animals with 0 or 1 weeks of prevascularization regardless of the Matrigel coating. Wider airway patency was observed in group M than group S. Group M showed migration of epithelium over the scaffold from 4 weeks after transplantation and complete coverage with epithelium at 12 weeks, whereas group S showed migration of the epithelium from 14 weeks and incomplete coverage with epithelium even at 24 weeks. CONCLUSION: This two-step method, utilizing the platysma as an in vivo bioreactor, may be a promising approach to achieve long-term survival and enhanced luminal patency. Matrigel coating on the scaffold had a synergistic effect on epithelial regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1732-1740, 2021.

Synthesis and characterization of dexamethasone-conjugated linear polyethylenimine as a gene carrier.

Linear polyethylenimine (25 kDa, LPEI25k) has been shown to be an effective non-viral gene carrier with higher transfection and lower toxicity than branched polyethylenimine (BPEI) of comparable molecular weight. In this study, dexamethasone was conjugated to LPEI25k to improve the efficiency of gene delivery. Dexamethasone is a synthetic glucocorticoid receptor ligand. Dexamethasone-conjugated LPEI25k (LPEI-Dexa) was evaluated as a gene carrier in various cells. Gel retardation assays showed that LPEI-Dexa completely retarded plasmid DNA (pDNA) at a 0.75:1 weight ratio (LPEI/pDNA). LPEI-Dexa had the highest transfection efficiency at a 2:1 weight ratio (LPEI-Dexa/DNA). At this ratio, the size of the LPEI-Dexa/pDNA complex was approximately 125 nm and the zeta potential was 35 mV. LPEI-Dexa had higher transfection efficiency than LPEI and Lipofectamine 2000. In addition, the cytotoxicity of LPEI-Dexa was much lower than that of BPEI (25 kDa, BPEI25k). In conclusion, LPEI-Dexa has a high transfection efficiency and low toxicity and can therefore be used for non-viral gene delivery.

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.

Detection and Assessment of α-Synuclein Oligomers in the Urine of Parkinson's Disease Patients.

BACKGROUND: α-Synuclein (α-syn) is a major component of Lewy bodies, a pathologic marker of Parkinson's disease (PD) in post-mortem studies. The use of α-syn as a practical PD biomarker has been investigated by numerous researchers. However, reports of differences in α-syn levels in biofluids, such as cerebrospinal fluid, plasma, and saliva, between PD patients and controls are inconsistent. Recently, the measurement of α-syn oligomer levels has emerged as a novel approach to diagnose PD. OBJECTIVE: Lysates and culture media from two different types of dopaminergic neuronal cells or urine samples from 11 non-PD and 21 PD patients were collected and analyzed. METHODS: We developed and performed an enzyme-linked immuno-absorbent assay (ELISA) to detect various oligomeric α-syn using distinct pairs of antibodies. RESULTS: We validated our ELISA using rotenone-induced alterations of α-syn levels in human dopaminergic neurons. Total urinary α-syn levels, measured using our ELISA method, showed no difference between PD and non-PD individuals, but a higher level of α-syn oligomer recognized by MJFR-14-6-5-2 in PD urine samples was observed. Levels of distinct oligomeric α-syn detected by ASyO5 were lower in PD urine samples. Three different α-syn ELISA results were analyzed with respect to the severity of PD, but only the correlation between total α-syn levels and PD index was significant. CONCLUSION: Our findings suggest that detection of distinct oligomeric formations of α-syn and measurement of their levels in urine might be feasible for use in PD diagnostics.

Messenger RNA/polymeric carrier nanoparticles for delivery of heme oxygenase-1 gene in the post-ischemic brain.

Ischemic stroke is a cerebrovascular disease caused by narrowed cerebral arteries. Thrombolytic agents such as tissue-plasminogen activators have been used for recanalization of the blood supply into the ischemic region. However, ischemia-reperfusion damage continues to increase the infarction volume. In this study, heme oxygenase-1 (HO1)-mRNA was delivered into the brain, using a non-viral carrier. Various non-viral carriers such as polyethylenimine (25 kDa, PEI25k), lipofectamine, dexamethasone-conjugated PEI2k (Dexa-PEI2k), deoxycholic acid-conjugated PEI2k (DA-PEI2k), and R3V6 peptides were evaluated as carriers of mRNA into the brain. Gene delivery assays showed that DA-PEI2k and lipofectamine had a higher mRNA delivery efficiency than the other carriers in Neuro2A cells in vitro and a rat brain in vivo. Cytotoxicity assays showed that lipofectamine had higher toxicity than DA-PEI2k. Therefore, DA-PEI2k was used for delivery of HO1-mRNA. Unlike plasmid DNA (pDNA), mRNA is expressed in the cytosol without nuclear translocation. This suggests that mRNA may have higher gene expression than pDNA, since the nuclear location of pDNA is an inefficient step. Indeed, in in vitro transfection assays, HO1-mRNA/DA-PEI2k had higher gene expression than HO1-pDNA/DA-PEI2k without induction of a pro-inflammatory cytokine. The therapeutic effects of HO1-mRNA delivery using DA-PEI2k were evaluated in the middle cerebral artery occlusion animal model after local injection. HO1-mRNA delivery had higher gene expression than HO1-pDNA delivery 24 h after the local injection. In addition, HO1-mRNA delivery reduced the infarct size more efficiently than HO1-pDNA delivery. The results suggest that the delivery of mRNA using DA-PEI2k may be useful for gene therapy of ischemic stroke.

Dexamethasone-conjugated polyethylenimine as an efficient gene carrier with an anti-apoptotic effect to cardiomyocytes.

BACKGROUND: Dexamethasone is a potent glucocorticoid with anti-inflammatory effects. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply the anti-apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was synthesized and evaluated as an anti-apoptotic gene carrier. METHODS: PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). The transfection efficiency and cytotoxicity of PEI-Dexa were evaluated by luciferase assay and the MTT assay. To evaluate the anti-apoptotic effect, PEI-Dexa/DNA complex was transfected into cells and the cells were treated with H(2)O(2). Cell viability and apoptosis level were measured by the MTT assay and caspase-3 assay, respectively. RESULTS: A transfection assay into H9C2 rat cardiomyocytes showed that PEI-Dexa had the highest transfection efficiency at an 8 : 1 weight ratio (PEI-Dexa/DNA). At this ratio, PEI-Dexa had higher transfection efficiency than high molecular polyethylenimine (PEI25K, 25 kDa) and PEI2K. In addition, the cytotoxicity of PEI-Dexa was lower than that of PEI25K. To evaluate the anti-apoptotic effect, PEI-Dexa/pSV-Luc or PEI2K/pSV-Luc was transfected into H9C2 cells and the cells were treated with H(2)O(2). PEI-Dexa was found to reduce caspase-3 activity and increase cell viability compared to PEI2K. Heme oxygenase-1 (HO-1) can protect ischemic cardiomyocytes from apoptosis. Therefore, pSV-HO-1 was cloned and transfected into H9C2 cells using PEI-Dexa. The cells transfected with PEI-Dexa/pSV-HO-1 complex had lower caspase-3 activity and higher viability than the cells transfected with PEI-Dexa/pSV-Luc complex after the H(2)O(2) treatment. CONCLUSIONS: PEI-Dexa is an efficient gene carrier with an anti-apoptotic effect and may be useful for anti-apoptotic gene therapy in combination with pSV-HO-1.

A comparison of non-viral vectors for gene delivery to pancreatic beta-cells: delivering a hypoxia-inducible vascular endothelial growth factor gene to rat islets.

Although non-viral vectors are relatively safe, they have very low gene transfection efficiency, especially in pancreatic islet cells. To provide information on the use of non-viral vectors for transfecting genes into pancreatic islet cells, a comparative evaluation of non-viral options was performed. In vitro experiments were used to compare the transfection efficiency of three classes of non-viral vectors: Effectene, polyethylenimine (PEI, 25 kDa) and hemagglutinating virus of Japan-envelope (HVJ-E), into insulinoma cells (INS-1) and rat islets. Vascular endothelial growth factor (VEGF) gene with hypoxia-inducible RTP801 promoter was delivered into rat islets with Effectene and VEGF secretion under hypoxia was measured in the culture media. Luciferase activity and GFP assays indicated that Effectene exhibited the highest transfection efficiency, and HVJ-E was not suitable for transfection into pancreatic beta-cells. The cytotoxicity of Effectene was found to be similar to that of 25-kDa PEI by 7-amino actinomycin D (7-AAD) flow cytometry and acridine orange/propidium iodide (AO/PI) assays. When RTP801 promoter-VEGF plasmid was delivered to rat islets with Effectene, VEGF secretion increased specifically in islets under hypoxia. In conclusion, Effectene showed higher gene-delivery efficiency for pancreatic islets compared with other classes of non-viral delivery systems and is promising as a gene delivery agent for pretransplant ex vivo gene therapy of islets.

Safety of office-based percutaneous injection laryngoplasty with calcium hydroxylapatite.

OBJECTIVE: To evaluate the safety of office-based calcium hydroxylapatite (CaHA) injection laryngoplasty via the cricothyroid approach through an analysis of all procedures performed over a period of 10 years at a single institution. METHODS: In total, 962 office-based CaHA injection laryngoplasty via the cricothyroid approach procedures were performed by a single physician at our institution between 2007 and 2016. From these, 955 procedures performed in 617 patients were included in our analysis. The medical records of all 617 patients were retrospectively reviewed. We classified all procedure-related complications according to the time of onset. Complications that occurred during the procedure were considered intraprocedural complications, whereas complications that developed within 1 week after injection and those that developed after 1 week and were recorded more than twice in the medical records were considered acute and delayed complications, respectively. Failed cases were categorized separately as failure. RESULTS: Five cases were failed (0.5%). Intraprocedural complications included superficial injection in eight cases (0.8%). Acute and delayed onset of dyspnea was observed in three (0.3%) and two (0.2%) cases, respectively. The incidence of failures and major complications requiring active intervention was 1.6%. CONCLUSION: Our findings suggest that office-based CaHA injection laryngoplasty via the cricothyroid approach is as safe as conventional transoral injection laryngoplasty. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:2361-2365, 2019.

Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs.

Polymeric nanosphere-mediated gene delivery may sustain the duration of plasmid DNA (pDNA) administration. In this study, poly(lactic-co-glycolic acid) (PLGA) nanospheres were evaluated as a gene carrier. The pDNA-loaded PLGA nanospheres were formulated with high encapsulation efficiency (87%). The nanospheres sustained release of pDNA for 11 days. The released pDNA maintained its structural and functional integrity. Furthermore, the PLGA nanospheres showed lower cytotoxicity than polyethylenimine (PEI) in vitro and in vivo. The nanospheres with vascular endothelial growth factor (VEGF) gene were injected into skeletal muscle of ischemic limb model, and gene expression mediated by the PLGA nanospheres with VEGF gene was compared to that of PEI/pDNA or naked pDNA in vivo. PLGA nanosphere/pDNA had significantly higher VEGF expression levels in comparison to PEI/pDNA and naked pDNA at 12 days after administration. In addition, gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA. These results indicated that PLGA nanosphere might be useful as a potential carrier for skeletal muscle gene delivery applications.

Mitochondria targeting delivery of nucleic acids.

BACKGROUND: Mitochondria are intracellular organelles involved in energy production, which play important roles in metabolism. Consequently, mutation in mitochondrial DNA may have adverse effects on the host organism. This hypothesis is supported by increasing number of reports that associate various diseases with the mutation of the mitochondrial genome. Therefore, new therapy methods for targeting mitochondria genome should be developed for the treatment of these diseases. OBJECTIVES: The current progress in mitochondrial targeting gene delivery is discussed and future direction is suggested. METHODS: Recent research progress in this field is briefly introduced, and successes and obstacles in research are discussed. RESULTS/CONCLUSION: Delivery of antisense DNA using lipophilic cation showed possible therapeutic effect in vitro. Delivery of tRNA is also another possible approach to correct tRNA mutations. However, research into the delivery of protein expression system using liposome and polymer has been very limited. The results suggest that more research is required to address the problems in mitochondrial targeting gene delivery. Here, we suggest 'multifunctional multilamella vesicular or multifunctional multi-vesicular (MMV)' for efficient mitochondrial targeting DNA delivery.

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.