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Topical treatment of various skin disorders requires drug absorption and penetration through the stratum corneum (SC) into the epidermis and dermis tissues. The use of nano-drug delivery systems including liposomes and lipid nanoparticles (SLNs) have been shown to facilitate SC penetration. The goal of this work was to study the impact of liposome sizes and the resulted drug distribution inside various skin tissue. All trans retinoic acid (ATRA) was used as the model drug and loaded into gel phase HSPC/CHOL/DSPE-PEG liposomes (lipo-ATRA) with sizes ranging from 80 nm to more than 300 nm. The percutaneous drug absorption process was monitored and analyzed. There were significant differences in percutaneous absorption and tissue distribution resulted from liposomes smaller than 100 nm and those bigger than 200 nm. Lipo-ATRA with a mean diameter of 83 nm can deliver the content to epidermis and dermis. But for 200 nm - 300 nm liposomes, the resulted epidermis and dermis ATRA levels were less than about one third, suggesting bigger liposomes had poor penetration through the brick and mortar structure of SC. Gel phase liposomes with sizes under 100 nm improved encapsulated drug absorption and distribution into the epidermis and dermis tissues. A size dependent mechanism for liposome penetration of the stratum corneum was proposed.

Nano-drug delivery systems are designed to contain surface ligands including antibodies for “active targeting”. The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the “targeting” property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2–22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications.

Malignant melanoma has shown increased incidence and high mortality rate in the last three decades. In this study, we investigated whether combination therapy with ch282-5 (a novel BH3 mimetic) and microwave hyperthermia could display synergistic antitumor effects against melanoma. Our results indicated that combination therapy reduced the viability and proliferation of melanoma cells. Through inhibiting the expression of anti-apoptotic proteins of Bcl-2 and IAP family and activating MAPK proteins, combined hyperthermia enhanced ch282-5-induced apoptosis. Combination therapy also synergistically disturbed the mTOR/p70S6k signaling pathway, which is important for cell survival and migration. Moreover, our results showed that combination therapy remarkably suppressed melanoma cell migration in vitro and significantly reduced experimental pulmonary metastasis in vivo. In conclusion, our results indicate that combination therapy with ch282-5 and hyperthermia has synergistic antitumor effects and provides a possible therapeutic strategy for advanced melanoma.

pH-responsive drug delivery systems have attracted great interest because of their potential use in antitumor therapies. Herein, we report a facile one-pot fabrication of a “host−metal−drug” coordination-bonding system in a mesostructured surfactant/silica hybrid for the pH-responsive drug delivery purpose. The mesostructure has been synthesized by self-assembly of nontoxic and biocompatible F127 Pluronic nonionic surfactant and silica source through a real liquid crystal templating route, in which F127 act as host molecules. Metal ions such as Zn, Cu, and Fe and drugs have been introduced simultaneously into the mesostructure synthesis systems, to form F127−metal−drug coordination-bonding architecture. The cleavage of the coordination bonds that are sensitive to variations in external pH gives rise to the release of the drug under weakly acidic conditions (pH 5.0−6.0). To assist in the release of drugs without significant binding capabilities, a vector has been designed to endow coordinately inert drugs ...

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers.

BACKGROUND: Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive. METHODS: We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests. RESULTS: We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001). CONCLUSIONS: Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target.

Although legumain has been found to be a prognostic factor in both breast cancer and colorectal cancer, its effects on gastric cancer are unknown. In this study, we investigated effects of legumain on gastric cancer and the correlation between legumain expression and prognosis of gastric cancer patients. SGC7901 cells were transduced with legumain cDNA (SGC7901-hLeg) for overexpression of legumain or with legumain shRNA to knock down legumain. In vitro tumor migration was examined by wound healing assay. Furthermore, a tumorigenicity and metastasis mouse model was used to examine legumain function in vivo; asparaginyl endopeptidase inhibitor (AEPI, an inhibitor of legumain) was injected to the mice (i.p.) to evaluate its therapeutic effect. Tissue microarray analysis from 112 gastric cancer patients was performed to evaluate the association between legumain expression and the cumulative survival time. Legumain was highly expressed in gastric cancer patients and some gastric cancer cell lines. Legumain promoted gastric cell migration in vitro and promoted gastric tumor growth and metastasis in vivo, and these effects were reversed by knockdown of legumain with shRNA or treated with AEPI. In gastric cancer clinical samples, legumain expression in tumor was significantly higher than in non-tumor and was negatively associated with the cumulative survival rate. In conclusion, legumain was highly expressed in gastric adenocarcinoma; legumain promoted gastric cancer tumorigenesis and metastasis in vitro and in vivo. Legumain expression in tumor was a poor prognostic factor for gastric cancer patients, and legumain could be a potential target molecule for gastric cancer therapy in clinic.

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25−Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4+Foxp3+ T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4+ effector T cells. The percentage of CD4+ T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

Quantum dots (QDs) have favorable physical and photochemical properties. In this work, we used QDs fluorescent nanoprobes to follow the migration of inflammatory cells from local tissue to draining lymph node in inflammation resolution. Electric pulse stimulation was used to establish inflammation model in mouse tibialis anterior. QDs injected to inflammatory tissue were found to aggregate and endocytosed by inflammatory cells. While in the draining lymph node, QDs mainly distributed in the T cell area. TEM and confocal observation showed that most of QDs in the draining lymph node were located in the endosomes of monocytes/macrophages. Our work shows that QDs can be used as fluorescent probes to follow migration of cells especially phagocytic cells. Thus QDs may be explored in application in immunology research.

Micro/nano-particle drug delivery systems are being developed for improving therapeutic effects. However, most studies can only evaluate the delivery properties by indirectly measuring the pharmacokinetic changes of the drugs. It had been difficult to directly assay the drug carrier status and distribution in vivo. We described here a method using intravital microscopy to visualize individual drug carriers traveling through blood vessels. The transportation and extravasation process of these particles were monitored. The in vivo distribution patterns were also confirmed by fluorescence microscopy studies of the various tissue samples.

Asparagine endopeptidase (AEP) has been shown to play important roles in innate immunity. Here, we show that AEP is a previously unrecognized regulator of autophagy in M2 macrophages. AEP interacted with PI3K p110α and suppressed PI3K activation on the endomembranes. AEP deficiency led to abnormal activation of PI3K, which converted phosphatidyinositol (4,5)-bisphosphate (PIP2) to phosphatidyinositol (3,4,5)-bisphosphate (PIP3) (PIP3). Decrease in PIP2 and increase in PIP3 resulted in impaired autophagic lysosome biogenesis and autolysosome clearance. Impaired autolysosome clearance in turn led to accumulation of NF-κB proteins, which promoted proinflammatory cytokine synthesis upon LPS stimulation. Moreover, AEP-/- mice showed significantly enhanced inflammation when stimulated with LPS or challenged with S. Japonicum eggs. Induction of autophagy with raramycin in AEP-/- mice and inhibition of autophagy with Bafilomaycin A1 in WT mice prevented and increased S. Japonicum eggs induced granulomas respectively. Thus, our data linked AEP to the immunosuppressive phenotype of M2 macrophages through promoting autophagy.

Electroporation can improve intramuscular DNA vaccination efficacy but the exact antigen presentation mechanism remains unclear. We reported here that a similar immuno-potentiation effect was also observed by stimulating the skeletal muscles with electric pulses (EP) a few days prior to DNA inoculation (EP + n days + DNA). The application of EP by itself activated proinflammatory chemokine genes and stress genes. It also triggered an influx of inflammatory monocytes/macrophages (MPs). After DNA inoculation, the plasmids were seen taken up by these inflammatory MPs, which migrated to the draining LNs subsequently. The antibody responses results were fast and strong. Furthermore, MPs isolated from the draining LNs of EP + n days + DNA treated mice were capable of stimulating Ag specific CD4+ T cell proliferation in vitro. Based on these observations, we proposed that the local inflammation resulted from EP treatment played an important role in facilitating antigen presentation of the DNA vaccines.

Electroporation has been shown to be an effective method to improve the efficiency of gene expression and the immunogenicity of DNA vaccines. In order to optimize the procedure and test for its efficacy in more clinically relevant large animal models, we examined the detailed immune responses in rhesus macaques after vaccination intramuscularly with electroporation using the plasmid encoding for HBV preS(2)-S antigen and an adjuvant plasmid encoding for hIL-2 and hIFN-gamma. Several important factors were examined, including the dose response relationships, the effect of various prime and boost regimens, and different combinations of electro-pulse parameters. The immune responses were closely followed for more than a year. The results showed that in rhesus macaques, electroporation can significantly enhance the immunogenicity of the DNA vaccines, resulting in greatly improved antibody responses as well as peptide-stimulated IFN-gamma producing T cell responses. In addition, we also reported the different antibody response behaviors resulted from different electro-pulse parameters. The detailed data would be useful to suggest possible optimization strategies for better DNA vaccine efficacy.

Gene delivery to skeletal muscles assisted by electroporation(EP) is a promising strategy for in vivo production of the therapeutic proteins. But the commonly used procedure required the application of electric pulses with voltages at above 200V/cm and durations at least 40 ms, which would result in several damages in the muscle and limited surviving cells expressing transgene. We reported here an optimization study of the various electric pulse parameters to reduce toxicity while maintaining transgene expression. In addition, we also found that the secreted transgene product level detected in serum samples may not correlate with the total gene expression level in muscles. Based on our data, we'd propose some less damaging electroporation parameters that may be useful for intramuscular gene delivery and therapeutic protein production

The immunogenecity of intramuscularly injected plasmid DNA vaccine can be greatly enhanced by in vivo electroporation. We investigated the possible mechanism of the immune potentiation effect. Severe tissue damage in TA muscles with broken perimysium and edomysium were observed after electroporation, which facilitated DNA distribution and cell permeation. There was a process of necrosis/apoptosis in the TA muscles as shown by the activation of several stress and toxicity related genes including Caspase-8, Caspase-1, MT2 and cytochrome P450. Many chemokines and chemokine receptors gene expression were also activated. The pathological changes and molecular level alteration of gene expression in the TA muscles recruited heavy infiltration of mononuclear cells, which were shown to distribute in the space between transgene (GFP) expressing muscle fibers. Macrophages were present in abundance in the muscles as early as 3 days after electroporation, and were observed through out our study to day 21. But only a few DC cells were present at day 14 and day 21. Based on those observations, we hypothesize that the infiltrating macrophages are responsible for cross-presentation of antigen from transfected muscle fibers to T cells. Indeed, we observed T cells activation in the draining LN by chemokines and receptors cDNA array, with enhanced expression of CXCR-1, CXCR-2, CCR-1, CCR-3, CCR-4, CCR-7, CCR-8, CCR-9 and Cmkbr1l2 genes. CD4+ and CD8+ T cells were observed to appear in the muscle at as early as day 5, peak between day 7 and day 14, but disappear at day 21 by immunohistochemistry analysis. Full recovery of the muscle fibers was found at day 28 after electroporation, with center nucleated regenerated muscle cells and eliminated inflammatory cell infiltrates.

We report here the successful use of anti-CD3 antibodies to modulate the immune responses after gene therapy. Five consecutive medium doses of anti-CD3 treatment concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic-level of FVIII gene expression in treated Hemophilia A mice. Moreover, repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells in treated mice and increased the frequency of CD4+CD25+FoxP3+ regulatory T cells (Tregs). Nevertheless, prior depletion of CD25+ T cells did not abrogated tolerance induction and adoptive transfer of CD4+CD25+ T cells of tolerized mice in early time points to syngeneic plasmid injected mice did not transfer the dominant tolerance. Furthermore, anti-CD3 treated mice could mount normal immune responses against both T-dependent and T-independent neo-antigens. Our data indicate that anti-CD3 immunomodulation can be a safe and effective strategy to prevent transgene-specific immune responses following gene therapy.

Formation of inhibitory antibodies is a significant problem encountered in the treatment of hemophilia by replacement therapy. Nonviral gene transfer of a factor VIII plasmid into hemophilia mice induced strong humoral responses through predominantly TH2 signals. The plasmid-treated mice produced persistent, high-level inhibitory antibody specifically against FVIII, representing a unique and useful model for testing various immunomodulation strategies. It was previously demonstrated by our group that transient immunosuppression by CTLA4-Ig and anti-CD40L (MR1) can prevent inhibitory antibody formation following nonviral gene transfer of FVIII plasmid into hemophilia A mice. In this study, we tested if blockade of inducible costimulator (ICOS)-ICOS ligand (ICOSL) pathway in combination with or without agents blocking other co-stimulatory pathways can modulate the immune response following gene therapy treatment. Three groups of mice (n=5/group) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection, and transient immunosuppressive regimens including anti-ICOS (8 treatment in 2 week period), combination of anti-ICOS (same dose) and CTLA4-Ig (2 treatment at day 0 and 2), and combination of anti-ICOS (same dose) and MR1 (5 treatment in 2 week period). 2 mice from anti-ICOS only group, 3 mice from combination treatment of anti-ICOS and CTLA4-Ig group, and 2 mice from combination treatment of anti-ICOS and MR1 group developed inhibitors at 2 weeks post treatment. The rest of the mice did not develop inhibitors. These results imply that neither synergistic nor additional modulation was achieved by combining CTLA4-Ig or MR1 with anti-ICOS compared to anti-ICOS alone. Subsequently a more frequent and longer anti-ICOS treatment (16 treatment in 4 week period) was administered in two separate groups of FVIII plasmid-treated mice (n=5 and 11 per group, respectively). All the treated mice did not produce inhibitory antibodies against FVIII and produced persistent, high-level (100–300 μg/ml) FVIII gene expression for at least 150 days (experimental period). The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro. Furthermore, higher population of CD4+CD25+ regulatory T cells were detected in peripheral blood in the tolerized mice compared to untreated and plasmid-treated mice. Adoptive transfer of CD4+ T cells isolated from tolerized mice is performed to test if these cells can protect the recipient mice from developing inhibitory antibodies against FVIII. Anti-ICOS treatment has the potential for a new immunomodulatory strategy for preventing the formation of inhibitory antibodies against FVIII following gene therapy.