HPMA copolymer-phospholipase C and dextrin-phospholipase A2 as model triggers for polymer enzyme liposome therapy (PELT).

'Polymer Enzyme Liposome Therapy' (PELT) is a two-step anticancer approach in which a liposomal drug and polymer-phospholipase conjugate are administered sequentially to target the tumour interstitium by the enhanced permeability and retention effect, and trigger rapid, local, drug release. To date, however, the concept has only been described theoretically. We synthesised two polymer conjugates of phospholipase C (PLC) and A2 (PLA2) and evaluated their ability to trigger anthracycline release from the clinically used liposomes, Caelyx® and DaunoXome®. N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-PLC and a dextrin-PLA2 were synthesised and their enzymatic activity characterised. Doxorubicin release from polyethyleneglycol-coated (PEGylated) Caelyx® was relatively slow (<20%, 60 min), whereas daunomycin was rapidly released from non-PEGylated DaunoXome® (∼87%) by both enzymes. Incubation with dextrin-PLA2 triggered significantly less daunomycin release than HPMA copolymer-PLC, but when dextrin-PLA2 was pre-incubated with α-amylase, the rate of daunomycin release increased. DaunoXome®'s diameter increased in the presence of PLA2, while Caelyx®'s diameter was unaffected by free or conjugated PLA2. Dextrin-PLA2 potentiated the cytotoxicity of DaunoXome® to MCF-7 cells to a greater extent than free PLA2, while combining dextrin-PLA2 with Caelyx® resulted in antagonism, even in the presence of α-amylase, presumably due to steric hindrance by PEG. Our findings suggest that in vivo studies to evaluate PELT combinations should be further evaluated.

Protective Effect of the Sulfated Agaran Isolated from the Red Seaweed Laurencia aldingensis Against Toxic Effects of the Venom of the Snake, Lachesis muta.

Snakebite is a serious occupational hazard affecting mainly rural populations of tropical and subtropical developing countries. Lachesis muta (Bushmaster) bites are extremely serious but are rarely reported in the literature. Bushmaster envenomings are characterized by intense local pain, edema, neurotoxicity, hypotension, local hemorrhage, and dramatic systemic alterations. Antivenom treatment has regularly been used for more than a century; however, it fails to neutralize local tissue damage and hemorrhage, leading to morbidity or disabilities in victims. Thus, the production and clinical use of antivenom must be improved. The present work characterizes, for the first time, a sulfated polysaccharide from the red seaweed, Laurencia aldingensis, including its neutralizing effect on some toxic activities of L. muta venom. Chemical and spectroscopic analyses showed that L. aldingensis produces sulfated agarans with the A-units partially C-2 sulfated or 6-O-methoxylated presetting the B-units in the cyclized (3,6-anhydro-α-L-galactose) or in the non-cyclized form (α-L-galactose). The latter is significantly substituted by sulfate groups on C-6. In vitro and in vivo assays showed that this sulfated agaran inhibited hemolysis, coagulation, proteolysis, edema, and hemorrhage of L. muta venom. Neutralization of hemorrhagic activity was also observed when the agaran was administered by different routes and after or before the venom injection. Furthermore, the agaran blocked the edema caused by a phospholipase A2 isolated from the L. muta venom. Experimental evidence therefore indicates that the sulfated agaran of L. aldingensis has potential to aid antivenom therapy of accidents caused by L. muta venom and may help to develop more effective antivenom treatments of snake bites in general.

Protective Effects of Intratracheally-Administered Bee Venom Phospholipase A2 on Ovalbumin-Induced Allergic Asthma in Mice.

Asthma is a common chronic disease characterized by bronchial inflammation, reversible airway obstruction, and airway hyperresponsiveness (AHR). Current therapeutic options for the management of asthma include inhaled corticosteroids and ß2 agonists, which elicit harmful side effects. In the present study, we examined the capacity of phospholipase A2 (PLA2), one of the major components of bee venom (BV), to reduce airway inflammation and improve lung function in an experimental model of asthma. Allergic asthma was induced in female BALB/c mice by intraperitoneal administration of ovalbumin (OVA) on days 0 and 14, followed by intratracheal challenge with 1% OVA six times between days 22 and 30. The infiltration of immune cells, such as Th2 cytokines in the lungs, and the lung histology, were assessed in the OVA-challenged mice in the presence and absence of an intratracheal administration of bvPLA2. We showed that the intratracheal administration of bvPLA2 markedly suppressed the OVA-induced allergic airway inflammation by reducing AHR, overall area of inflammation, and goblet cell hyperplasia. Furthermore, the suppression was associated with a significant decrease in the production of Th2 cytokines, such as IL-4, IL-5, and IL-13, and a reduction in the number of total cells, including eosinophils, macrophages, and neutrophils in the airway.

Efficacy of a therapeutic treatment using gas-filled microbubble-associated phospholipase A2 in a mouse model of honeybee venom allergy.

BACKGROUND: Venom immunotherapy is efficient to desensitize people suffering from insect sting allergies. However, the numerous injections required over several years and important risks of severe side reactions complicate the widespread use of immunotherapy. In the search for novel approaches to blunt the overwhelming pro-allergic Th2 response, we evaluated the therapeutic efficacy of a treatment based on a denatured form of the major allergen, phospholipase A2, associated with microbubbles (PLA2denat -MB) in a mouse model of honeybee venom allergy. METHODS: Antibodies measured by ELISA, T-cell responses assessed by CFSE-based proliferation assays and ELISA, and basophil degranulation were examined after PLA2denat -MB-based therapeutic treatment of sensitized mice. Mice were challenged with a lethal dose of PLA2 to evaluate protection against anaphylaxis. RESULTS: Therapeutic subcutaneous administration of two different PLA2denat -MB formulations, in contrast to PLA2denat alone, reduced allergic symptoms and protected all mice from anaphylaxis-mediated death after allergen challenge. At the functional level, the use of PLA2denat decreased IgE-mediated basophil degranulation as compared to the native form of the allergen. In comparison with PLA2denat alone, both PLA2denat -MB formulations decreased allergen-specific Th2 CD4 T-cell reactivity. At the mechanistic level, PLA2denat -MB containing 20% palmitic acid and PEG induced PLA2-specific IgA and increased Foxp3(+) Treg frequencies and TGF-ß production, whereas the formulation bearing 80% palmitic acid triggered the production of IFN-γ, IgG2a, and IgG3. CONCLUSIONS: In contrast to conventional PLA2 subcutaneous immunotherapy, the therapeutic administration of PLA2-MB treatment to mice that already had established allergy to PLA2 protects all subsequently challenged animals.

Prophylactic immunization of mice with phospholipase A2-loaded gas-filled microbubbles is protective against Th2-mediated honeybee venom allergy.

BACKGROUND: People suffering from honeybee venom allergy can be treated by venom immunotherapy, which consists in the subcutaneous injection of increasing doses of allergen extracts over a period of 3-5 years. Such a procedure is time-consuming, and the risks of severe side reactions are important. Approaches based on the use of novel adjuvants to blunt pro-allergic Th2-type immune responses represent a sound alternative. OBJECTIVES: In this study, we evaluated in a mouse model of honeybee venom allergy the protection induced by the prophylactic use of the major allergen phospholipase A2 (PLA2) associated with microbubbles (MB). METHODS: Antibody (Ab) and T cell responses, as detected by ELISA and CFSE-based proliferation assays, were first examined after prophylactic immunization of CBA/J mice with PLA2-MB, and second after sensitization with native PLA2. Mice were eventually challenged with a lethal dose of PLA2 to assess protection against anaphylaxis. RESULTS: Prophylactic immunization with PLA2-MB induced PLA2-specific IgG and IgA Ab, triggered the production of IFN-γ and IL-10 and the differentiation of PLA2-specific Foxp3(+) Treg. Immunized/sensitized mice displayed the following: (1) increased titres of potent blocking IgG1, IgG2a and IgG3 Ab, (2) both reduced allergen-specific T cell proliferation and Th2-type cytokine production and (3) elevated frequencies of specific Foxp3(+) Treg and increased production of TGF-ß, as compared to naïve/sensitized animals. Immunomodulation correlated with reduced signs of anaphylaxis after allergen challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate the ability of PLA2-MB to prophylactically protect mice against subsequent sensitization and death-inducing PLA2 challenge for up to 4 months, revealing so far unravelled immunomodulatory properties of MB. These data, combined with the safe use of MB as contrast agents for in situ imaging in humans, render them an immunotherapeutic agent of great interest for further evaluation.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

Phospholipase A(2)-susceptible liposomes of anticancer double lipid-prodrugs.

A novel approach to anticancer drug delivery is presented based on lipid-like liposome-forming anticancer prodrugs that are susceptible to secretory phospholipase A(2) (sPLA(2)) that is overexpressed in several cancer types. The approach provides a selective unloading of anticancer drugs at the target tissues, as well as circumvents the necessity for "conventional" drug loading. In our attempts to improve the performance of the liposomes in vivo, several PEGylated and non-PEGylated liposomal formulations composed of a retinoid prodrug premixed with the sPLA(2)-hydrolyzable DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared. Besides favorably modifying the physicochemical properties of the liposomes, the incorporation of DPPC and PEG-lipids in the liposomes should substantially enhance the enzymatic activity, as concluded from literature. In addition, one can reap benefits from the presumed permeability enhancing effect of the liberated fatty acids and lysolipids. The size distribution of the prepared liposomes as well as their phase behavior, enzymatic hydrolysis, and cytotoxicity, in the presence and absence of sPLA(2), were determined. The liposomes were around 100nm in diameter and in the gel/fluid coexistence region at 37°C. The enzymatic hydrolysis of the prodrug was pronouncedly accelerated upon the premixing with DPPC, and the hydrolysis was further enhanced by PEGylation. Interestingly, the faster hydrolysis of the prodrug and the released fatty acids and lysolipids from DPPC did not improve the cytotoxicity of the mixture; the effect of combining the prodrug with DPPC was additive and not synergistic. The data presented here question the significance of the permeability enhancing effects claimed for fatty acids and lysolipids at the target cell membrane, and whether these effects can be achieved using physiologically achievable concentrations of fatty acids and lysolipids.

Polysaccharide nanogel gene delivery system with endosome-escaping function: Co-delivery of plasmid DNA and phospholipase A2.

We developed a novel gene delivery system capable of endosome disruption using a polysaccharide-based cationic nanogel composed of a hexadecyl group-bearing cationic cycloamylose nanogel (C16-catCA nanogel) and phospholipaseA(2) (PLA(2)) to hydrolyze membrane phospholipids. C16-catCA nanogel formed nanoparticles with PLA(2) and pDNA by hydrophobic and electrostatic interactions. Both pDNA and PLA(2) were effectively internalized into cells by the C16-catCA nanogel. In addition, the pDNA expression level was enhanced when complexed with specific concentrations of PLA(2). PLA(2) complexed with C16-catCA nanogel also showed a similar hemolytic activity against red blood cells to that observed using native PLA(2). These results suggest that the C16-catCA nanogel/PLA(2) complex possesses membrane disruption ability when delivered into cells and triggers the subsequent release of pDNA from the endosome to the cytoplasm. This is the first report of co-delivery of pDNA and PLA(2) using the same carrier to achieve effective gene delivery.