This study was performed to explore other potential mechanisms underlying hemolysis in addition to pore-formation of tentacle extract (TE) from the jellyfish Cyanea capillata. A dose-dependent increase of hemolysis was observed in rat erythrocyte suspensions and the hemolytic activity of TE was enhanced in the presence of Ca²⁺, which was attenuated by Ca²⁺ channel blockers (Diltiazem, Verapamil and Nifedipine). Direct intracellular Ca²⁺ increase was observed after TE treatment by confocal laser scanning microscopy, and the Ca²⁺ increase could be depressed by Diltiazem. The osmotic protectant polyethylenglycol (PEG) significantly blocked hemolysis with a molecular mass exceeding 4000 Da. These results support a pore-forming mechanism of TE in the erythrocyte membrane, which is consistent with previous studies by us and other groups. The concentration of malondialdehyde (MDA), an important marker of lipid peroxidation, increased dose-dependently in rat erythrocytes after TE treatment, while in vitro hemolysis of TE was inhibited by the antioxidants ascorbic acid—Vitamin C (Vc)—and reduced glutathione (GSH). Furthermore, in vivo hemolysis and electrolyte change after TE administration could be partly recovered by Vc. These results indicate that lipid peroxidation is another potential mechanism besides pore-formation underlying the hemolysis of TE, and both Ca²⁺ channel blockers and antioxidants could be useful candidates against the hemolytic activity of jellyfish venoms.

Our previous studies demonstrated that tentacle extract (TE) from the jellyfish, Cyanea capillata, could cause a dose-dependent increase of systolic blood pressure, which seemed to be the result of direct constriction of vascular smooth muscle (VSM). The aim of this study is to investigate whether TE could induce vasoconstriction in vitro and to explore its potential mechanism. Using isolated aorta rings, a direct contractile response of TE was verified, which showed that TE could induce concentration-dependent contractile responses in both endothelium-intact and -denuded aortas. Interestingly, the amplitude of contraction in the endothelium-denuded aorta was much stronger than that in the endothelium-intact one, implying that TE might also bring a weak functional relaxation in addition to vasoconstriction. Further drug intervention experiments indicated that the functional vasodilation might be mediated by nitric oxide, and that TE-induced vasoconstriction could be attributed to calcium influx via voltage-operated calcium channels (VOCCs) from the extracellular space, as well as sarcoplasmic reticulum (SR) Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (IP₃R), leading to an increase in [Ca²⁺]_c, instead of activation of the PLC/DAG/PKC pathway or the sympathetic nerve system.

We first identified and characterized a novel peroxiredoxin (Prx), designated as CcPrx4, from the cDNA library of the tentacle of the jellyfish Cyanea capillata. The full-length cDNA sequence of CcPrx4 consisted of 884 nucleotides with an open reading frame encoding a mature protein of 247 amino acids. It showed a significant homology to peroxiredoxin 4 (Prx4) with the highly conserved F-motif (⁹³FTFVCPTEI¹⁰¹), hydrophobic region (²¹⁷VCPAGW²²²), ¹⁴⁰GGLG¹⁴³ and ²³⁹YF²⁴⁰, indicating that it should be a new member of the Prx4 family. The deduced CcPrx4 protein had a calculated molecular mass of 27.2 kDa and an estimated isoelectric point of 6.3. Quantitative real-time PCR analysis showed that CcPrx4 mRNA could be detected in all the jellyfish tissues analyzed. CcPrx4 protein was cloned into the expression vector, pET-24a, and expressed in Escherichia coli Rosetta (DE3) pLysS. Recombinant CcPrx4 protein was purified by HisTrap High Performance chelating column chromatography and analyzed for its biological function. The results showed that the purified recombinant CcPrx4 protein manifested the ability to reduce hydrogen peroxide and protect supercoiled DNA from oxidative damage, suggesting that CcPrx4 protein may play an important role in protecting jellyfish from oxidative damage.

To investigate the hematologic changes and the activities of jellyfish venoms other than hemolytic and cardiovascular toxicities, the acute toxicity of tentacle-only extract (TOE) from the jellyfish Cyanea capillata was observed in mice, and hematological indexes were examined in rats. The median lethal dose (LD₅₀) of TOE was 4.25 mg/kg, and the acute toxicity involved both heart- and nervous system-related symptoms. Arterial blood gas indexes, including pH, PCO₂, HCO₃⁻, HCO₃std, TCO₂, BEecf and BE (B), decreased significantly. PO₂ showed a slight increase, while SO₂c (%) had no change at any time. Na⁺ and Ca²⁺ decreased, but K⁺ increased. Biochemical indexes, including LDH, CK, CK-MB, ALT, AST and sCr, significantly increased. Other biochemical indexes, including BUN and hemodiastase, remained normal. Lactic acid significantly increased, while glucose, Hct% and THbc showed slight temporary increases and then returned to normal. These results on the acute toxicity and hematological changes should improve our understanding of the in vivo pathophysiological effects of TOE from C. capillata and indicate that it may also have neurotoxicity, liver toxicity and muscular toxicity in addition to hemolytic and cardiovascular toxicities, but no kidney or pancreatic toxicity.

Nanodrug delivery systems have been used extensively to improve the tumor-targeting ability and reduce the side effects of anticancer drugs. In this study, nanomicelles responsive to dual stimuli were designed and developed as drug carriers for delivering doxorubicin (DOX). The hydrophobic group of the nanomicelles was composed of the photosensitizer protoporphyrin IX (PpIX) and the disulfide bond-containing alpha-lipoic acid (LA); the hydrophilic group was made up of the nuclear localization signal (NLS, CGGGPKKKRKVGG) peptide with a lysine linker. Furthermore, anionic cyclo-γ-polyglutamic acid (cyclo-γ-PGA) was coated on the surface of the cationic micelles to construct a multifunctional drug delivery system (NLS-LA-PpIX-DOX@cyclo-γ-PGA). Cyclo-γ-PGA, as a biological coating material, notably improved the stability of the cationic micelles by reducing nonspecific reactions with anionic groups. Additionally, the cyclo-γ-PGA coating mediated active tumor targeting and enhanced the cellular uptake of micelles via the γ-glutamyl transpeptidase (GGT) pathway. The integrated micelles not only achieved photochemical internalization (PCI) and photodynamic therapy (PDT) via light-activated reactive oxygen species (ROS) but also realized controlled intracellular drug release via the glutathione (GSH)-responsive disulfide-bond cleavage. As a result, NLS-LA-PpIX-DOX@cyclo-γ-PGA exhibited excellent synergistic chemo-photodynamic antitumor activity and fewer side effects than other therapies both in vitro and in vivo. In conclusion, this new dual-responsive drug delivery system (NLS-LA-PpIX-DOX@cyclo-γ-PGA) with improved stability and enhanced tumor-targeting ability may facilitate the development of high-efficiency and low-toxicity nanotherapeutic approaches.