Characterization of in vitro neural functional connectivity on a neurofluidic device.

Understanding the mechanism of functional connectivity in neural system is of great benefit to lot of researches and applications. Microfluidics and microelectrode arrays (MEAs) have been frequently utilized for in vitro neural cultures study. However, there are few studies on the functional connectivity of neural cultures grown on a microfluidic chip. It is intriguing to unveil the influences of microfluidic structures on in vitro neuronal networks from the perspective of functional connectivity. Hence, in the present study, a device was established, which comprised a microfluidic chamber for cell growth and a MEA substrate for recording the electrophysiological response of the neuronal networks. The network topology, neural firing rate, neural bursting rate and network burst frequency were adopted as representative characteristics for neuronal networks analysis. Functional connectivity was estimated by means of cross-covariance analysis and graph theory. The results demonstrated that the functional connectivity of the in vitro neuronal networks formed in the microchannel has been apparently reinforced, corresponding to improve neuronal network density and increased small-worldness.

Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial.

BACKGROUND: Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. METHODS: Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). FINDINGS: Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4-36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3-34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3-37·8]; p=0·007). INTERPRETATION: VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. FUNDING: Inovio Pharmaceuticals.

Facile synthesis of MoS2@red phosphorus heterojunction for synergistically photodynamic and photothermal therapy of renal cell carcinoma.

The therapy of the clear cell renal cell carcinoma (ccRCC) is crucial for the human healthcare due to its easy metastasis and recurrence, as well as resistance to radiotherapy and chemotherapy. In this work, we propose the synthesis of MoS2@red phosphorus (MoS2@RP) heterojunction to induce synergistic photodynamic and photothermal therapy (PDT/PTT) of ccRCC. The MoS2@RP heterojunction exhibits enhanced spectra absorption in the NIR range and produce local heat-increasing under the NIR laser irradiation compared with pure MoS2 and RP. The high photocatalytic activity of the MoS2@RP heterojunction contributes to effective transferring of the photo-excited electrons from the RP to MoS2, which promotes the production of various types of radical oxygen species (ROS) to kill the ccRCC cells. After the NIR irradiation, the MoS2@RP can effectively induce the apoptosis in the ccRCC cells through localized hyperthermia and the generation of ROS, while exhibiting low cytotoxicity towards normal kidney cells. In comparison to MoS2, the MoS2@RP heterojunction shows an approximate increase of 22 % in the lethality rate of the ccRCC cells and no significant change in toxicity towards normal cells. Furthermore, the PDT/PTT treatment using the MoS2@RP heterojunction effectively eradicates a substantial number of deep-tissue ccRCC cells in vivo without causing significant damage to major organs. This study presents promising effect of the MoS2@RP heterojunction-based photo-responsive therapy for effective ccRCC treatment.

Personalized prediction of mortality in patients with acute ischemic stroke using explainable artificial intelligence.

BACKGROUND: Research into the acute kidney disease (AKD) after acute ischemic stroke (AIS) is rare, and how clinical features influence its prognosis remain unknown. We aim to employ interpretable machine learning (ML) models to study AIS and clarify its decision-making process in identifying the risk of mortality. METHODS: We conducted a retrospective cohort study involving AIS patients from January 2020 to June 2021. Patient data were randomly divided into training and test sets. Eight ML algorithms were employed to construct predictive models for mortality. The performance of the best model was evaluated using various metrics. Furthermore, we created an artificial intelligence (AI)-driven web application that leveraged the top ten most crucial features for mortality prediction. RESULTS: The study cohort consisted of 1633 AIS patients, among whom 257 (15.74%) developed subacute AKD, 173 (10.59%) experienced AKI recovery, and 65 (3.98%) met criteria for both AKI and AKD. The mortality rate stood at 4.84%. The LightGBM model displayed superior performance, boasting an AUROC of 0.96 for mortality prediction. The top five features linked to mortality were ACEI/ARE, renal function trajectories, neutrophil count, diuretics, and serum creatinine. Moreover, we designed a web application using the LightGBM model to estimate mortality risk. CONCLUSIONS: Complete renal function trajectories, including AKI and AKD, are vital for fitting mortality in AIS patients. An interpretable ML model effectively clarified its decision-making process for identifying AIS patients at risk of mortality. The AI-driven web application has the potential to contribute to the development of personalized early mortality prevention.

Influence of resilience on depression among nurses in clean operating departments: The mediating effect of life satisfaction.

BACKGROUND: A clean operating room is an important part of surgical and critical treatment in hospitals. The workload is substantial, the pace is rapid, and the working environment is intense; therefore, nurses who work in clean operating rooms are constantly challenged, which can lead to anxiety, depression, and other mental health issues. Life satisfaction and resilience are important factors that ensure mental health. Therefore, exploring the mediating role of life satisfaction in the influence of resilience on depression among nurses in clean operating rooms can help improve nursing services and teamwork. AIM: To explore the mediating effect of satisfaction on the influence of resilience on depression among nurses in a clean operating department. METHODS: From April to November 2022, 196 nurses from the Department of Clean Operating at Harbin Medical University Cancer Hospital participated in this study. Participants were selected using convenience sampling. Participants' gender, age, marital status, position, length of service, personal monthly income, daily working hours, employment status, and professional title were collected, and the Connor-Davidson resilience scale, satisfaction with life scale, and self-rating depression scale were used to evaluate resilience, life satisfaction, and depression. The researchers conducted professional training in advance, introduced the research methods to the participants before the investigation, and explained the study's significance and purpose. Surveys were distributed and collected on-site. Each questionnaire took 30 min to complete. RESULTS: The average scores for life satisfaction, resilience, and depression were 3.13 (± 0.28), 4.09 (± 0.78), and 56.21 (± 8.70), respectively. The correlation between resilience and depression was negative (r = -0.829, P < 0.01). Life satisfaction was positively related to resilience (r = 0.855, P < 0.01) and negatively related to depression (r = -0.778, P < 0.01). The relationship between resilience and depression was partially mediated by life satisfaction. The value of the mediating effect was -6.853 (26.68% of the total effect). CONCLUSION: Life satisfaction partially mediates the link between resilience and depression among nurses in clean operating departments.

Hexarelin alleviates apoptosis on ischemic acute kidney injury via MDM2/p53 pathway.

INTRODUCTION: Hexarelin exhibits significant protection against organ injury in models of ischemia/reperfusion (I/R)-induced injury (IRI). Nevertheless, the impact of Hexarelin on acute kidney injury (AKI) and its underlying mechanism remains unclear. In this study, we investigated the therapeutic potential of Hexarelin in I/R-induced AKI and elucidated its molecular mechanisms. METHODS: We assessed the protective effects of Hexarelin through both in vivo and in vitro experiments. In the I/R-induced AKI model, rats were pretreated with Hexarelin at 100 µg/kg/d for 7 days before being sacrificed 24 h post-IRI. Subsequently, kidney function, histology, and apoptosis were assessed. In vitro, hypoxia/reoxygenation (H/R)-induced HK-2 cell model was used to investigate the impact of Hexarelin on apoptosis in HK-2 cells. Then, we employed molecular docking using a pharmmapper server and autodock software to identify potential target proteins of Hexarelin. RESULTS: In this study, rats subjected to I/R developed severe kidney injury characterized by tubular necrosis, tubular dilatation, increased serum creatinine levels, and cell apoptosis. However, pretreatment with Hexarelin exhibited a protective effect by mitigating post-ischemic kidney pathological changes, improving renal function, and inhibiting apoptosis. This was achieved through the downregulation of conventional apoptosis-related genes, such as Caspase-3, Bax and Bad, and the upregulation of the anti-apoptotic protein Bcl-2. Consistent with the in vivo results, Hexarelin also reduced cell apoptosis in post-H/R HK-2 cells. Furthermore, our analysis using GSEA confirmed the essential role of the apoptosis pathway in I/R-induced AKI. Molecular docking revealed a strong binding affinity between Hexarelin and MDM2, suggesting the potential mechanism of Hexarelin's anti-apoptosis effect at least partially through its interaction with MDM2, a well-known negative regulator of apoptosis-related protein that of p53. To validate these findings, we evaluated the relative expression of MDM2 and p53 in I/R-induced AKI with or without Hexarelin pre-administration and observed a significant suppression of MDM2 and p53 by Hexarelin in both in vivo and in vitro experiments. CONCLUSION: Collectively, Hexarelin was identified as a promising medication in protecting apoptosis against I/R-induced AKI.

Rosiglitazone attenuates development of polycystic kidney disease and prolongs survival in Han:SPRD rats.

Although pioglitazone, a PPAR-gamma (peroxisome-proliferator-activated receptor-gamma) agonist, has been shown to prolong survival in two rapidly progressive pkd1 (polycystic kidney disease 1)-knockout mice models through disparate mechanisms, these studies lacked data on therapeutic potential and long-term safety because of a short observation period. In the present study, we have used another potent PPAR-gamma agonist, rosiglitazone, to treat Han:SPRD rats, a slowly progressive ADPKD (autosomal dominant PKD) animal model, and confirmed that short-term treatment was able to delay the progression of kidney cysts and protect renal function, which may relate to down-regulating the abnormally activated beta-catenin signalling pathway and its anti-inflammatory and anti-fibrosis effects. Long-term administration significantly prolonged the survival of Han:SPRD rats. Moreover, early therapy in rats with normal renal function had a better outcome than delayed therapy, while initiating therapy in rats with mild impaired renal function still protected renal function. The efficacy of rosiglitazone depended on continuous drug administration; withdrawal of the drug caused accelerated deterioration of renal function in effectively treated rats and shortened their survival to an untreated state. Long-term administration led to cardiac enlargement, probably due to rosiglitazone-mediated sodium re-absorption. In conclusion, these results indicate that rosiglitazone was able to effectively delay the progression of kidney disease and protect renal function in Han:SPRD rats, but its adverse effect of inducing cardiac enlargement should also be monitored closely.

Resolvin D1 Protects Against Ischemia/Reperfusion-Induced Acute Kidney Injury by Increasing Treg Percentages via the ALX/FPR2 Pathway.

AIMS: To evaluate whether Resolvin D1 attenuates ischemia/reperfusion-induced (IRI) acute kidney injury (AKI) via affecting Tregs. MATERIALS AND METHODS: The IRI-AKI mouse model was established, and RvD1 was injected into the mouse tail vein. Further, the renal function, histological changes, injury markers and serum cytokines were detected at 24 and 72 h after IRI. Flow cytometry was used to categorize regulatory T cells (Tregs) in the spleen and kidney. Treg cells were stripped with the anti-CD25 antibody blocker PC61 to assess its role in the protective effect of RvD1 on IRI mice. CD4+ T cells were obtained from spleen monocytes by magnetic bead sorting and differentiated into induced Treg (iTreg) cells. The effect of RvD1 on iTreg cell differentiation was observed in vitro. In addition, neutralizing antibodies against the orphan receptor G-protein-coupled receptor 32 (anti-GPR32) and LXA4 receptor (anti-ALX/FPR2), both RvD1 receptor blockers, were used to evaluate the effect of RvD1 on iTreg cell differentiation. Boc-1, an ALX/FPR2 receptor inhibitor, was administered via the tail vein to observe its effects on the ameliorative efficacy of RvD1 in IRI-AKI mice in vivo. RESULTS: In vivo, RvD1 increased Treg percentages, alleviated renal tubular injury and reduced the serum levels of IFN-γ, TNF-α and IL-6 in IRI-AKI mice, while PC61 depleted the number of Tregs and reversed the protective effects of RvD1. In vitro, RvD1 induced the generation of iTregs. Importantly, preincubation with anti-ALX/FPR2 neutralizing antibodies but not with anti-GPR32 neutralizing antibodies, abrogated the enhancement activity of RvD1 on iTregs. In addition, in vivo blockade of the receptor ALX/FPR2 by Boc-1 reversed the beneficial effects of RvD1 on the splenic and kidney Treg percentages, renal tubular injury and serum IFN-γ, TNF-α, and IL-6 levels. CONCLUSION: Our study demonstrates that RvD1 protects against IRI-AKI by increasing the percentages of Tregs via the ALX/FPR2 pathway.

Critical Role of Cysteine-Rich Protein 61 in Mediating the Activation of Renal Fibroblasts.

OBJECTIVE: To explore the expression of cysteine-rich protein 61 (Cyr61) in ischemic renal fibrosis and the role of Cyr61 in mediating the activation of renal fibroblasts. METHODS: (1) The rat model of renal fibrosis was established after ischemia-reperfusion acute renal injury (IR-AKI). We detected the renal function by biochemical test, evaluated the fibrosis by Masson staining, and detected the expression of Cyr61 by western blotting. (2) Bioinformatics technique was adopted to analyze the expression of Cyr61 in activated renal fibroblasts. (3) Normal rat kidney fibroblast cells (NRK-49F cells) with over-expression of Cyr61 (Cyr61+) and low-expression of it (Cyr61--) were established by plasmid transfection. Then part of the cells were activated by TGF-ß1 and NRK-49F cells were divided into control group, activated group, Cyr61+/Cyr61-- group and Cyr61+/Cyr61-- activated group. The expression of Cyr61 and fibrosis related factors (Col1α1, Col3α1, MMP9, and MMP13) were ascertained by PCR and western blotting. Cell proliferation was discovered by CCK8 method, cell cycle was analyzed by flow cytometry, and the transcription of cell senescence related factors (P53, P21, Rb, and P16) were ascertained by PCR method. RESULTS: (1) In the process of fibrosis after IR-AKI, the area of collagen fiber was most obviously at AKI 1W, while the Cyr61 protein was at the lowest level at AKI 1W. (2) Gene chip analysis showed that the expression of Cyr61 was decreased in renal fibroblasts after IR. (3) Compared with control group, Cyr61+ group expressed less Col1α1 or Col3α1, as well as more MMP9 and MMP13. At the same time, the proliferation of Cyr61+ group decreased and cells in G1 phases increased with more transcription of P53, P21, and Rb (all P < 0.05). Compared with activated group, the results of Cyr61+ activated group were similar to the above. The above effects of low expression group were just the opposite. In addition, there was no difference in the transcription of P16 among these groups (P > 0.05). CONCLUSION: Cyr61 may not only inhibit the fibrotic phenotype of fibroblasts, but may also inhibit proliferation by promoting fibroblasts arrest in G1 phase through the P53/P21/Rb interrelated cell senescence pathway, subsequently affecting the process of ischemic renal fibrosis.

Preclinical evaluation of multi antigenic HCV DNA vaccine for the prevention of Hepatitis C virus infection.

Direct-acting antiviral treatment for hepatitis C virus (HCV) infection is costly and does not protect from re-infection. For human and chimpanzees, recovery from acute HCV infection correlates with host CD4+ and CD8+ T cell responses. DNA plasmids targeting the HCV non-structural antigens NS3, NS4, and NS5, were previously reported to induce robust and sustained T cell responses in mice and primates. These plasmids were combined with a plasmid encoding cytokine IL-28B, together named as VGX-6150. The dose-dependent T cell response and safety of VGX-6150 administered intramuscularly and followed by electroporation was assessed in mice. Immune responses plateaued at 20 µg/dose with IL-28B demonstrating significant immunoadjuvant activity. Mice administered VGX-6150 at 40, 400, and 800 µg given either as a single injection or as 14 injections given bi-weekly over 26 weeks showed no vaccine related changes in any clinical parameter compared to placebo recipients. There was no evidence of VGX-6150 accumulation at the injection site or in any organ 1 month following the 14th vaccination. Based on these studies, the approximate lethal dose (ALD) exceeds 800 µg/dose and the NOAEL was 800 µg/dose in mouse. In conclusion, VGX-6150 appears safe and a promising preventive vaccine candidate for HCV infection.

Augmentation of cellular and humoral immune responses to HPV16 and HPV18 E6 and E7 antigens by VGX-3100.

We have previously demonstrated the immunogenicity of VGX-3100, a multicomponent DNA immunotherapy for the treatment of Human Papillomavirus (HPV)16/18-positive CIN2/3 in a phase 1 clinical trial. Here, we report on the ability to boost immune responses with an additional dose of VGX-3100. Patients completing our initial phase 1 trial were offered enrollment into a follow on trial consisting of a single boost dose of VGX-3100. Data show both cellular and humoral immune responses could be augmented above pre-boost levels, including the induction of interferon (IFN)γ production, tumor necrosis factor (TNF)α production, CD8+ T cell activation and the synthesis of lytic proteins. Moreover, observation of antigen-specific regulation of immune-related gene transcripts suggests the induction of a proinflammatory response following the boost. Analysis of T cell receptor (TCR) sequencing suggests the localization of putative HPV-specific T cell clones to the cervical mucosa, which underscores the putative mechanism of action of lesion regression and HPV16/18 elimination noted in our double-blind placebo-controlled phase 2B trial. Taken together, these data indicate that VGX-3100 drives the induction of robust cellular and humoral immune responses that can be augmented by a fourth "booster" dose. These data could be important in the scope of increasing the clinical efficacy rate of VGX-3100.

Mutation analysis of autosomal dominant polycystic kidney disease genes in Han Chinese.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2. The complexity of these genes, particularly PKD1, has complicated genetic screening, though recent advances have provided new opportunities for amplifying these genes. In the Han Chinese population, no complete mutational analysis has previously been conducted across the entire span of PKD1 and PKD2. Here, we used single-strand conformation polymorphism (SSCP) analysis to screen the entire coding sequence of PKD1 and PKD2 in 85 healthy controls and 72 Han Chinese from 24 ADPKD pedigrees. In addition to 11 normal variants, we identified 17 mutations (12 in PKD1 and 5 in PKD2), 15 of which were novel ones (11 for PKD1 and 4 for PKD2). We did not identify any seeming mutational hot spots in PKD1 and PKD2. Notably, we found several disease-associated C-T or G-A mutations that led to charge or hydrophobicity changes in the corresponding amino acids. This suggests that the mutations cause conformational alterations in the PKD1 and PKD2 protein products that may impact the normal protein functions. Our study is the first report of screenable mutations in the full-length PKD1 and PKD2 genes of the Han Chinese, and also offers a benchmark for comparisons between Caucasian and Han ADPKD pedigrees and patients.

[Expression of PKD1 and PKD2 transcripts and proteins and its significance in different types of kidney tissues and kidney lines].

OBJECTIVE: To investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines. METHODS: Immunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines. RESULTS: Coordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01). CONCLUSIONS: Similar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

Influenza A vaccines using linear expression cassettes delivered via electroporation afford full protection against challenge in a mouse model.

Alternative DNA vaccine constructs such as fully synthetic linear expressing cassettes (LECs) offer the advantage of accelerated manufacturing techniques as well as the lack of both antibiotic resistance genes and bacterial contaminants. The speed of manufacture makes LEC technology a possible future vaccination strategy for pandemic influenza outbreaks. Previously, we reported on a novel concept of DNA delivery to dermal tissue by a minimally invasive electroporation (EP) surface device powered using low voltage parameters. This device allows electroporation without penetration of electrodes into the skin. In addition to enhancing the delivery of traditional plasmid DNA vaccines, this device may also offer a safe, tolerable and efficient method to administer LECs. To assess immunogenicity and efficacy of EP-enhanced LEC delivery in mice, we designed and tested two influenza antigens in the form of LEC constructs delivered using the newly developed surface dermal EP device. Strong CTL and antibody responses were induced by the LEC versions of the DNA vaccine. When challenged with A/Canada/AB/RV1532/2009 viruses, mice immunized with LEC encoding the M2 and NP antigens recovered faster than naïve or mice immunized ID without EP. Mice immunized with equal-molar doses of LEC encoding the M2 and NP antigens demonstrated 100% survival following a lethal (100× LD50) challenge of the heterologuos and highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04). These results suggest that influenza DNA vaccines based on LEC technology combined with the surface delivery platform are capable of fully protecting mice in a lethal challenge and the LEC based DNA constructs may serve as viable vaccine candidates.

Optimized in vivo transfer of small interfering RNA targeting dermal tissue using in vivo surface electroporation.

Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner.

Optimization of electroporation-enhanced intradermal delivery of DNA vaccine using a minimally invasive surface device.

In vivo electroporation (EP) is an efficient nonviral method for enhancing DNA vaccine delivery and immunogenicity in animals and humans. Intradermal delivery of DNA vaccines is an attractive strategy because of the immunocompetence of skin tissue. We have previously reported a minimally invasive surface intradermal EP (SEP) device for delivery of prophylactic DNA vaccines. Robust antibody responses were induced after vaccine delivery via surface EP in several tested animal models. Here we further investigated the optimal EP parameters for efficient delivery of DNA vaccines, with a specific emphasis on eliciting cellular immunity in addition to robust humoral responses. In a mouse model, using applied voltages of 10-100 V, transgene expression of green fluorescent protein and luciferase reporter genes increased significantly when voltages as low as 10 V were used as compared with DNA injection only. Tissue damage to skin was undetectable when voltages of 20 V and less were applied. However, inflammation and bruising became apparent at voltages above 40 V. Delivery of DNA vaccines encoding influenza virus H5 hemagglutinin (H5HA) and nucleoprotein (NP) of influenza H1N1 at applied voltages of 10-100 V elicited robust and sustained antibody responses. In addition, low-voltage (less than 20 V) EP elicited higher and more sustained cellular immune responses when compared with the higher voltage (above 20 V) EP groups after two immunizations. The data confirm that low-voltage EP, using the SEP device, is capable of efficient delivery of DNA vaccines into the skin, and establishes that these parameters are sufficient to elicit both robust and sustainable humoral as well as cellular immune responses without tissue damage. The SEP device, functioning within these parameters, may have important clinical applications for delivery of prophylactic DNA vaccines against diseases such as HIV infection, malaria, and tuberculosis that require both cellular and humoral immune responses for protection.