Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

Oxygen-producing proenzyme hydrogels for photodynamic-mediated metastasis-inhibiting combinational therapy.

Photodynamic therapy (PDT) has provided a promising approach for the treatment of solid tumors, while the therapeutic efficacy is often limited due to the hypoxic tumor microenvironment, resulting in tumor metastasis. Herein, we report an oxygen-producing proenzyme hydrogel (OPeH) with photoactivatable enzymatic activity for PDT enabled metastasis-inhibiting combinational therapy of breast cancer. This OPeH based on alginate is composed of protoporphyrin IX (PpIX) conjugated manganese oxide (MnO2) nanoparticles, which act as both the photosensitizer and oxygen-producing agent, and singlet oxygen (1O2)-responsive proenzyme nanoparticles. In the hypoxic and acidic tumor microenvironment, MnO2 can generate 1O2 to promote PpIX-mediated PDT with an amplified 1O2 generation efficiency, which also triggers the cleavage of 1O2-responsive linkers and cascade activation of proenzymes for cancer cell death. This combinational therapy upon photoactivation not only greatly inhibited the tumor growth, but also suppressed lung metastasis in a mouse xenograft breast tumor model, which is impossible in the case of PDT alone. This study thus provides a proenzyme hydrogel platform with photoactivatable activity for metastasis-inhibiting cancer therapy with high efficacy and safety.

Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy.

Cancer cells immersed in inherent oxidative stress are more vulnerable to exogenous oxidative damages than normal cells. Reactive oxygen species (ROS)-mediated oxidation therapy preferentially aggravating tumor oxidative stress to disrupt redox homeostasis, has emerged as an effective and specific anticancer treatment. Herein, following an ingenious strategy of "broaden sources and reduce expenditure", we designed a versatile tumor-specific oxidative stress nanoamplifier enabling economized photodynamic therapy (PDT), to achieve synergistic oxidative stress explosion for superior oxidation therapy. Methods: Cinnamaldehyde (CA) as a therapeutic ROS generator was first conjugated to hyaluronic acid (HA) through acid-labile hydrazone bond to synthesize tailored amphiphilic HA@CA conjugates, which could surprisingly self-assemble into uniform nanofibers in aqueous media. Photosensitizer protoporphyrin (PpIX) was efficiently encapsulated into HA@CA nanofibers and transformed HA@CA nanofibers to final spherical HA@CAP. Results: With beneficial pH-responsiveness and morphology transformation, improved bioavailability and selective tumor accumulation, HA@CAP combining ROS-based dual chemo/photodynamic treatment modalities could induce cytotoxic ROS generation in a two-pronged approach to amplify tumor oxidative stress, termed "broaden sources". Moreover, utilizing CA-induced H2O2 production and cascaded Fenton reaction in mitochondria to consume intracellular overloaded Fe(II), HA@CAP could skillfully block endogenic heme biosynthesis pathway on site to restrain undesired elimination of PpIX for economized PDT, termed "reduce expenditure". Both in vitro and in vivo results demonstrated the superior antitumor performance of HA@CAP. Conclusion: This study offered an inspiring strategy of "broaden sources and reduce expenditure" to specifically boost tumor oxidative stress for reinforced oxidation therapy.

Optically controlled hybrid metamaterial of plasmonic spiky gold inbuilt graphene sheets for bimodal imaging guided multimodal therapy.

The development of multifunctional molecular diagnostic platforms for the concordant visualization and treatment of diseases with high sensitivity and resolution has recently become a crucial strategy in cancer management. Thus, engineering functional metamaterials with high therapeutic and imaging capabilities to elucidate diseases from their morphological behaviors to physiological mechanisms is an unmet need in the current scenario. Here, we report the design of a unique hybrid plasmonic nanoarchitecture for targeted multiple phototherapies of breast cancer by simultaneous real-time monitoring through fluorescence and surface-enhanced Raman scattering (SERS) techniques. The nanoframework consisted of plasmonic gold-graphene hybrids tethered with folic acid-ligated chitosan-modified photosensitizer (PpIX) to afford target-specific localized photothermal and photodynamic therapy. The hybrid vehicle also served as an excellent nanocarrier for the efficient loading and stimuli-responsive release of the chemotherapeutic drug doxorubicin (DOX) to enhance the therapeutic efficacy, thereby forming a trimodal nanomedicine against cancer. The cytotoxic effects induced by the cumulative action of the triplet therapeutic tools were visualized through both fluorescence and SERS imaging channels. Moreover, it also generated synchronized therapeutic effects resulting in the effective regression of tumor volume without propagating any toxic effects to other organs of the animals. Taken together, by virtue of strong light-matter interactions, the nanoprobe showed enhanced photoadsorption, which facilitated amplified light-reactive therapeutic and imaging efficacies along with targeted and enhanced chemotherapy, both in vitro and in vivo, which may offer promising outcomes in clinical research.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas.

BACKGROUND: The usefulness of 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is intensely discussed. However, data about kinetics of 5-ALA and protoporphyrin (Pp) IX in meningiomas are lacking. METHODS: As the first study so far, we performed longitudinal intraoperative real-time ex situ measurements of fluorescence intensity and PpIX concentrations during FGS of ten benign and two atypical meningiomas. Kinetics were subsequently compared with data from 229 glioblastomas. RESULTS: Spectroscopy revealed fluorescence (median 2945.65 a.u.) and PpIX accumulation (median 18.31 µg/ml) in all 43 analyzed samples. Fluorescence intensity (2961.50 a.u. vs 118.41 a.u.; p < .001) and PpIX concentrations (18.72 µg/ml vs .98 µg/ml; p < .001) were higher in samples with (N = 30) than without (N = 2) visible intraoperative tumor fluorescence. ROC curve analyses revealed a PpIX cut-off concentration of 3.85 µg/ml (AUC = .992, p = .005) and a quantitative fluorescence cut-off intensity of 286.73 a.u. (AUC = .983, p = .006) for intraoperative visible tumor fluorescence. Neither fluorescence intensity (p = .356) nor PpIX (p = .631) differed between atypical and benign meningiomas. Fluorescence and PpIX peaked 7-8 h following administration of 5-ALA. Meningiomas displayed a higher fluorescence intensity (p = .012) and PpIX concentration (p = .005) than glioblastomas 5-6 h after administration of 5-ALA. Although fluorescence was basically maintained, PpIX appeared to be cleared faster in meningiomas than in glioblastomas. CONCLUSIONS: Kinetics of PpIX and fluorescence intensity differ between meningiomas and glioblastomas in the early phase after 5-ALA administration. Modification of the timing of drug administration might impact visibility of intraoperative fluorescence and helpfulness of FGS and should be investigated in future analyses.

Investigation of the emission spectra and cytotoxicity of TiO2 and Ti-MSN/PpIX nanoparticles to induce photodynamic effects using X-ray.

BACKGROUND: Photodynamic therapy (PDT) has been recognized as an effective method for cancer treatment; however, it suffers from limited tissue penetration depth. X-rays are ideal excitation sources for activating self-lighting nanoparticles that can penetrate through deep tumor tissues and convert the X-rays to visible light. In this study, Ti-MSN/PpIX nanoparticles for X-ray induced photodynamic therapy was synthesized. Preparation, characterization, and emission spectrum of Ti-MSN/PpIX nanoparticles as well as PDT activity and toxicity of the nanoparticles on HT-29 cell line were investigated. METHODS: Firstly, mesoporous silica nanoparticles (MSN) were synthesized through sol-gel method. Then, TiO2 and PpIX were loaded in MSN. Next, the emission spectra of TiO2, Ti-MSN, and Ti-MSN/PpIX nanoparticles, while activated by X-ray (6 MVp), were recorded. In addition, viability of cells after treatment by Ti-MSN/PpIX nanoparticles and X-ray irradiation was studied. RESULTS: SEM, TEM and FESEM images of the spherical composite nanoparticles showed that their dimensions were changed by incorporating Ti and organic compound of PpIX. Two-dimensional hexagonal structure with d100-spacing was about 3.5 nm with particle sizes of 70-110 nm. The optical characteristics of TiO2 nanoparticles showed strong emission lines, which effectively overlapped with the absorption wavelengths of protoporphyrin IX (PpIX). Cellular experiments showed Ti-MSN/PpIX nanoparticles have proper biocompatibility, however, after X-ray irradiation, significant decrease of cell viability in the presence of the nanoparticles was observed. CONCLUSION: The presented X-PDT method could enhance RT efficacy and is enable that allows for reducing X-ray dose exposure to healthy tissues to overcome radio-resistant tumors.

A Pharmacologic "Stress Test" for Assessing Select Antioxidant Defenses in Patients with CKD.

BACKGROUND AND OBJECTIVES: Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves. DESIGN: , setting, participants, & measurementsA total of 18 healthy volunteers and 24 participants with stage 3 CKD (n=12; eGFR 30-59 ml/min per 1.73 m2) or stage 4 CKD (n=12; eGFR 15-29 ml/min per 1.73 m2) were injected once with SnPP (9, 27, or 90 mg). Plasma and/or urinary antioxidant proteins were measured at baseline and for up to 4 days post-SnPP dosing. Kidney safety was gauged by serial measurements of BUN, creatinine, eGFR, albuminuria, and four urinary AKI biomarkers (kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, cystatin C, and N-acetyl glucosaminidase). RESULTS: Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (r=-0.85 to -0.95). All four proteins manifested statistically significant dose- and time-dependent elevations after SnPP injection. However, marked intersubject differences were observed. p21 responses to high-dose SnPP and HO-1 responses to low-dose SnPP were significantly suppressed in participants with CKD versus healthy volunteers. SnPP was well tolerated by all participants, and no evidence of nephrotoxicity was observed. CONCLUSIONS: SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.

Protein assembled nano-vehicle entrapping photosensitizer molecules for efficient lung carcinoma therapy.

The efficiency of drug depends not only on its potency but also on its ability to reach the target sites in preference to non-target sites. In this regard, protein assembled nanocarrier is the most promising strategy for intracellular anti-cancer drug delivery. The key motive of this study is to fabricate biocompatible protein assembled nanocarrier conjugated photosensitizer system for stimuli-responsive treatment of lung carcinoma. Here, we have synthesized a unique nanohybrid of protein assembled gold nanoparticles (AuNPs), attaching a model photosensitizer, Protoporphyrin IX (PpIX) to the protein shell of the nanoparticles (NPs) imparting an ideal drug-carrier nature. Photo-induced alteration in hydrodynamic diameter suggests structural perturbation of the nanohybrid which in terms signifies on-demand drug delivery. The drug release profile has been further confirmed by using steady-state fluorescence experiments. AuNP-PpIX showed excellent anti-cancer efficiency upon green light irradiation on lung adenocarcinoma cell line (A549) through intracellular reactive oxygen species (ROS) generation. The cellular morphological changes upon PDT and internalization of nanohybrid were monitored using confocal laser scanning microscope. This anti-cancer effect of nanohybrid was associated with apoptotic pathway which was confirmed in the flow cytometric platform. The developed nanomedicine is expected to find relevance in clinical anti-cancer PDT models in the near future.

Near-infrared light-triggered degradable hyaluronic acid hydrogel for on-demand drug release and combined chemo-photodynamic therapy.

In this study, an injectable and near-infrared (NIR) light-triggered ROS-degradable hyaluronic acid hydrogel platform was developed as localized delivery vehicle for photosensitizer protophorphyrin IX (PpIX) and anticancer drug doxorubicin (DOX), to achieve superior combined chemo-photodynamic therapy with light-tunable on-demand drug release. The in situ-forming hydrogel fabricated readily via the formation of dynamic covalent acylhydrazone bonds could efficiently prevent severe self-quenching effect of water-insoluble PpIX due to the covalent binding, leading to localized enhanced photodynamic therapy (PDT). Moreover, the extensive ROS generated by the hydrogel under NIR light irradiation could not only realize efficient PDT effect, but also cleave the ROS-cleavable small molecule crosslinker, inducing the desirable degradation of hydrogel and subsequent on-demand DOX release for cascaded chemotherapy. The developed versatile hyaluronic acid hydrogels have tunable properties, excellent biocompatibility, biodegradability and exhibit outstanding therapeutic effects in both in vitro cellular experiments and in vivo antitumor studies.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair.

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Ultraviolet radiation exposure during daylight Photodynamic Therapy.

BACKGROUND: Daylight photodynamic therapy (dPDT) is an effective treatment for field-change actinic keratoses (AK), with similar efficacy to conventional PDT but lower patient pain scores. Whilst AK occur consequent to chronic solar ultraviolet (UV) exposure, paradoxically solar visible radiation is used during PDT. OBJECTIVES: To investigate the nature and levels of UV exposure, both erythemal UV and UVA, occurring during dPDT. METHODS: Four years of solar erythemally effective UV (UVE) irradiance, UVA irradiance and illuminance data were obtained from Pubic Health England for 12 locations. For a standard 2 h treatment period, the data were converted into standard erythemal doses (SEDs), UVA dose and protoporphyrin-IX (PpIX)-weighted dose from UVE irradiance, UVA irradiance and illuminance respectively. These three parameters were compared ascertaining the UV exposure received during dPDT. RESULTS: Analysis of UV exposure during dPDT showed a UK maximum average UVE exposure of 8.2 SED at Camborne (PpIX dose 23.4 J cm-2). Treatment earlier in the day reduces average UV exposure (Camborne 5.2 SED, PpIX dose 18.2 J cm-2), whilst PpIX dose achieves threshold during winter months (Camborne, November, 0.8 SED, PpIX dose 7.1 J cm-2). Cyprus and Gibraltar (with high UV exposure during dPDT) experience a maximum of 14.3 SED and 12.9 SED, with respective PpIX doses of 36.1 J cm-2 and 35.1 J cm-2, in June. UVA exposure is also presented for comparison. CONCLUSION: Therapeutically effective dPDT doses can be achieved at times of the day and year when UV exposure is minimal.

Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid-mediated Protoporphyrin IX by Iron Chelator Deferoxamine.

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron-dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA-PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA-PpIX fluorescence in tumor cells with lower FECH activity (MDA-MB-231, Hs 578T) than in tumor cells with higher FECH activity (MDA-MB-453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.

Photodynamic therapy for actinic keratosis of the forehead and scalp with the Aktilite CL 128: Is there a cut-off value for PpIX-weighted irradiance for effective treatment?

BACKGROUND/PURPOSE: Photodynamic therapy (PDT) is an established treatment for actinic keratosis (AK). Among the approved protocols in Europe, the most widely used involves irradiation with the Aktilite CL 128 (C-PDT). We aimed to assess the heterogeneity of irradiance over the treatment area when using C-PDT. We also investigated whether there is a cut-off value for protoporphyrin IX (PpIX)-weighted irradiance that may predict the treatment outcome of C-PDT. METHODS: An Ophir PD300 photodiode sensor connected to an Ophir Laser Star power meter was used to measure the irradiance delivered to 114 AKs of the scalp and forehead of 19 patients treated with C-PDT. The PpIX-weighted irradiances were deduced and cross-referenced with the complete responses at 3 months. RESULTS: From the measured irradiances ranging from 0.25 to 60 mW/cm2 (average: 31.94 mW/cm2 ), a standard deviation of 17.17 mW/cm2 was computed. Irradiance heterogeneity over the treatment area during C-PDT was demonstrated. The 66/114 AKs with a complete response at 3 months (57.89%) received a mean PpIX-weighted irradiance of 0.52 mW/cm2 vs 0.56 mW/cm2 for the resistant 48/114 AKs (42.11%). No significant effect of PpIX-weighted irradiance on the complete response at 3 months was found (odds ratio for a 0.1-unit change, 0.96; 95% confidence interval, 0.83 to 1.10; P = 0.53). Therefore, no cut-off value for PpIX-weighted irradiance that predicts treatment outcome could be identified. CONCLUSIONS: A device enabling homogeneous irradiation at a lower irradiance than the Aktilite CL 128 may therefore be suitable. This lower irradiance may further increase the treatment tolerance by patients.

Mitochondria and plasma membrane dual-targeted chimeric peptide for single-agent synergistic photodynamic therapy.

Mitochondria and cell membrane play important roles in maintaining cellular activity and stability. Here, a single-agent self-delivery chimeric peptide based nanoparticle (designated as M-ChiP) was developed for mitochondria and plasma membrane dual-targeted photodynamic tumor therapy. Without additional carrier, M-ChiP possessed high drug loading efficacy as well as the excellent ability of producing reactive oxygen species (ROS). Moreover, the dual-targeting property facilitated the effective subcellular localization of photosensitizer protoporphyrin IX (PpIX) to generate ROS in situ for enhanced photodynamic therapy (PDT). Notably, plasma membrane-targeted PDT would enhance the membrane permeability to improve the cellular delivery of M-ChiP, and even directly disrupt the cell membrane to induce cell necrosis. Additionally, mitochondria-targeted PDT would decrease mitochondrial membrane potential and significantly promote the cell apoptosis. Both in vitro and in vivo investigations indicated that this combinatorial PDT in mitochondria and plasma membrane could achieve the therapeutic effect maximization with reduced side effects. The single-agent self-delivery system with dual-targeting strategy was demonstrated to be a promising nanoplatform for synergistic tumor therapy.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-ß, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

Early heme oxygenase 1 induction delays tumour initiation and enhances DNA damage repair in liver macrophages of Mdr2-/- mice.

Multi drug resistance protein 2 knockout mice (Mdr2-/-) are a mouse model of chronic liver inflammation and inflammation-induced tumour development. Here we investigated the kinetics of early heme oxygenase 1 (HO-1) induction on inflammation, tumour development, and DNA damage in Mdr2-/- mice. HO-1 was induced by intraperitoneal injection of cobalt protoporphyrin IX (CoPP) twice weekly for 9 consecutive weeks. Immediately after HO-1 induction, liver function improved and infiltration of CD4+ and CD8+ T cells was reduced. Furthermore, we observed increased p38 activation with concomitant reduction of Cyclin D1 expression in aged Mdr2-/- mice. Long-term effects of HO-1 induction included increased CD8+ T cell infiltration as well as delayed and reduced tumour growth in one-year-old animals. Unexpectedly, DNA double-strand breaks were detected predominantly in macrophages of 65-week-old Mdr2-/- mice, while DNA damage was reduced in response to early HO-1 induction in vivo and in vitro. Overall, early induction of HO-1 in Mdr2-/- mice had a beneficial short-term effect on liver function and reduced hepatic T cell accumulation. Long-term effects of early HO-1 induction were increased CD8+ T cell numbers, decreased proliferation as wells as reduced DNA damage in liver macrophages of aged animals, accompanied by delayed and reduced tumour growth.

Plasma membrane-anchorable photosensitizing nanomicelles for lipid raft-responsive and light-controllable intracellular drug delivery.

The past decade has witnessed a growing number of nanoparticulate drug delivery systems for cancer treatment. However, insufficient cellular uptake by cancer cells and the undesirable endo/lysosomal entrapment of internalized therapeutic drugs remain the "Achilles heel" of many developed nanoagents. Here, we develop a novel lipid raft-responsive and light-controllable polymeric drug for efficient cytosolic delivery of photosensitizers. Conjugating a photosensitizer protoporphyrin IX (PpIX) to a polyethylene glycol-cholesterol polymer affords the amphiphilic drug (denoted as Chol-PEG-PpIX) that forms micelles in aqueous solutions. The Chol-PEG-PpIX with two hydrophobic units (cholesterol and PpIX) showed robust binding to plasma membranes and enabled significant cellular uptake via two pathways: (1) cholesterol moiety triggered the lipid raft-mediated endocytosis of Chol-PEG-PpIX with minimized endo/lysosomal trafficking after internalization; (2) the membrane-bound PpIX acted as a light-controlled trigger and can augment the permeability of plasma membranes upon laser irradiation, allowing the rapid influx of extracellular Chol-PEG-PpIX within 5 min. For systemic drug delivery, Chol-PEG-PpIX was anchored on the surface of liposomes via in situ membrane modification, which substantially avoided nonspecific binding of Chol-PEG-PpIX to red blood cells during circulation. Besides, the Chol-PEG-PpIX-anchored liposomes exhibited enhanced in vivo fluorescence, reduced liver uptake, prolonged tumor retention, and effective tumor ablation by photodynamic therapy. This work illustrates a new strategy for direct and efficient cytosolic delivery of photosensitizers, which may hold great promise in cancer therapy.

Stabilized tetraether lipids based particles guided prophyrins photodynamic therapy.

Photodynamic therapy (PDT) that involves ergonomically delivered light in the presence of archetypical photosensitizer such as Protoporphyrin IX (PpIX) is a time-honored missile strategy in cancer therapeutics. Yet, the premature release of PpIX is one of the most abundant dilemma encounters the therapeutic outcomes of PDT due to associated toxicity and redistribution to serum proteins. In this study, ultrastable tetraether lipids (TELs) based liposomes were developed. PpIX molecules were identified to reside physically in the monolayer; thereby the inherent π-π stacking that leads to aggregation of PpIX in aqueous milieu was dramatically improved. TEL29.9 mol% and TEL62mol% based liposomes revealed PpIX sustained release diffusion pattern from spherical particles as confirmed by converged fitting to Baker & Lonsdale model. Stability in presence of human serum albumins, a key element for PDT accomplishment was emphasized. The epitome candidates were selected for vascular photodynamic (vPDT) in in-Ovo chick chorioallantoic membrane. Profoundly, TEL62mol% based liposomes proved to be the most effective liposomes that demonstrated localized effect within the irradiated area without eliciting quiescent vasculatures damages. Cellular photodynamic therapy (cPDT) revealed that various radiant exposure doses of 134, 202, 403 or 672 mJ.cm-2 could deliberately modulate the photo-responses of PpIX in TEL-liposomes.

Hemopexin promotes angiogenesis via up-regulating HO-1 in rats after cerebral ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) is a critical pathophysiological change of ischemic stroke. Heme-oxygenase-1 (HO-1) is a rate-limiting enzyme of eliminating excessive free heme by combining with hemopexin (HPX), a plasma protein contributing to alleviating infarct size due to ischemia stroke. This study was to investigate whether HPX could improve angiogenesis after cerebral ischemia-reperfusion via up-regulating HO-1. METHODS: Rats were randomly divided into five groups: sham, MCAO, MCAO + Vehicle, MCAO + HPX and MCAO + HPX + protoporphyrin IX (ZnPPIX, an HO-1 inhibitor). Cerebral I/R was induced by MCAO. Saline, vehicle, HPX and HPX + ZnPPIX were respectively given to MCAO group, MCAO + Vehicle group, MCAO + HPX group and MCAO + HPX + ZnPPIX group at the moment after reperfusion by intracerebroventricular injection. Neurological behavioral scores(NBS) was assessed at 24 h and 7d after I/R. Real-time polymerase chain reaction (RT-PCR) was used to analyze the mRNA level of HO-1. Angiogenesis in penumbra area was assessed by immunofluorescence detection at 7d after I/R. Serum endothelial nitric oxide synthase (eNOS) was assessed by enzyme linked immunosorbent assay (ELISA) at 24 h and 7d after I/R. RESULTS: Compared with sham group, the NBS and the mRNA levels of HO-1 at 24 h and 7d after I/R in MCAO group decreased notably (P < 0.05), the new vessel density in ischemia penumbra increased notably at 7d after I/R (P < 0.05), the serum eNOS level increased at 24 h and 7d after I/R (P < 0.05). MCAO group and MCAO + Vehicle group showed no significant differences (P > 0.05). In the MCAO + HPX group, compared with MCAO + Vehicle group, the NBS and the mRNA levels of HO-1 increased drastically at 24 h and 7d after I/R (P < 0.05), the new vessel density in ischemia penumbra increased significantly at 7d after I/R (P < 0.05), the serum eNOS level at 24 h and 7d after I/R ascended notably (P < 0.05). Compared with MCAO + HPX group, the NBS assessment, new vessel density and serum eNOS level decreased at corresponding time points after I/R in MCAO + HPX+ ZnPPIX group (P < 0.05). CONCLUSION: HPX can promote angiogenesis after cerebral ischemia-reperfusion injury in rats via up-regulating HO-1.

Avaliação da zinco protoporfirina (ZPP) em ruminantes domésticos / Zinc protoporphyrin (ZPP) evaluation in domestic ruminants

Nos sistemas de criação de ruminantes, a anemia crônica pode levar a grandes prejuízos econômicos, sendo decorrente da deficiência de ferro no organismo. Quando este se torna indisponível para ser incorporado à hemoglobina, forma-se um composto denominado zinco protoporfirina (ZPP), que pode ser um marcador precoce para a anemia, útil, portanto, para seu diagnóstico. Porém, para a utilidade dessa mensuração, é necessário que se conheçam os valores normais de ZPP para cada espécie. Assim, foram utilizados 30 bezerros, 30 caprinos e 30 ovinos, todos saudáveis, nos quais foram mensurados esses valores. Essa mensuração foi determinada em amostras de sangue refrigeradas, coletadas com EDTA, obtendo-se valores em hemácias não lavadas e lavadas. A lavagem visou à eliminação de substâncias interferentes nessas medidas. A média da ZPP nas amostras não lavadas foi de 80,9µmol ZPP/mol de heme nos bezerros; 55,09µmol ZPP/mol de heme nos caprinos e 73,76µmol ZPP/mol de heme nos ovinos. Após a lavagem, os valores foram 61,4µmol ZPP/mol de heme; 43,92µmol ZPP/mol de heme e 59,36µmol ZPP/mol de heme, nos bezerros, caprinos e ovinos, respectivamente. Devido à praticidade da técnica, essa pode ser empregada para a detecção precoce da anemia ferropriva, sendo recomendada a prévia lavagem das hemácias.(AU)

In ruminant breeding systems, chronic anemia can lead to economic losses, resulting from iron deficiency in the organismo. When iron is unavailable for incorporation into hemoglobin, a compound called zinc protoporphyrin (ZPP) is formed, may be an early marker for anemia and is useful for its diagnosis. However, for this measurement to be useful, it is necessary to know the normal values for the species. Therefore, 30 calves, 30 goats and 30 sheep, all of them healthy, to standardize the values were used. This measurement was determined on refrigerated blood samples collected with EDTA, obtaining values in red blood cells not washed and washed. The washing aimed at the elimination of interfering substances in these measures. The mean of the ZPP in the unwashed samples was 80,9µmol ZPP/mol of heme in calves; 55,09µmol ZPP/mol of heme in goats and 73,76µmol ZPP/mol of heme in sheep. After washing, the values were 61,4µmol ZPP/mol of heme; 43,92µmol ZPP/mol of heme e 59,36µmol ZPP/mol of heme, in calves, goats and sheep, respectively. Due to its practicality, the techniquecan be used for the early detection of iron deficiency anemia, recommending the previous lavage of the red blood cells.(AU)