| Part ⅠAs a representative of the phylum Cnidaria,jellyfish is a group of marine invertebrates which are widely distributed throughout most of the world’s oceans.Jellyfish venom is a complex polypeptide/protein mixture stored in the specialized stinging organelles(nematocysts)that are mainly localized in the tentacles.However,variations in the prey and geographical distribution of different jellyfish have made their nematocysts morphologically and functionally heterogeneous such that different species of jellyfish may produce quite different toxic effects.Thus,in the first part of this paper,two major blooming species in China,Cyanea capillata(C.capillata)and Nemopilema nomurai(N.nomurai)were chosed as research subjects to make comprehensive comparative studies on their sting-related toxic components.Since the diversity of toxins is mainly generated in the tentacles and nematocysts,we first used fresh jellyfish tentacles for RNA-Seq analysis to build high-quality tentacles transcriptome.Then,two penetrant nematocysts,which are used for prey capture and defense,were isolated from C.capillata and N.nomurai and the venoms inside were extracted for proteomics analysis.The main results are as follows:1.In our previous studies,we have constructed a cDNA library of the tentacle from C.capillata.In this paper,RNA-seq and de novo assembly were used to construct an N.nomurai tentacle transcriptome to provide predicted protein sequences for proteomic study.A total of 52,449,308 clean reads corresponding to more than 4.72 billion clean nucleotides were generated.These short reads were then de novo assembled into 265,993 contigs with an average length of 297 bases using the Trinity program.Then the contigs were connected,and a total of 118,243 unigenes were obtained.The assembled unigenes were annotated using the Blastx algorithm(E-value≤10-5)against a set of public databases,including the Nr,Swiss-Prot,Pfam,KOG,GO,and KEGG databases.A total of 15,927 unigenes were found in all the databases(Nr,Swiss-Prot,KOG,GO and KEGG).In the KOG annotation,a total of 25,733 unigenes were grouped into 25 functional categories.For the GO annotation,26,933 unigenes were annotated and grouped according to biological process,cellular component and molecular function.With regard to the KEGG annotation,19,423 unigenes were enriched in diverse categories,including organismal systems,metabolism,genetic information processing,environmental information processing and cellular processes.2.The nematocysts from N.nomurai and C.capillata were purified using an optimized method.Both light and scanning electron microscopic techniques were used to elucidate morphological differences between the nematocysts of N.nomurai and C.capillata that might be related to the diversity of the putative toxin proteins in their venoms.Light microscopy revealed that N.nomurai nematocysts were quite different from C.capillata nematocysts in shape and size.Taxonomically,isorhiza-type nematocysts were the predominant type in N.nomurai,which possess the ability to penetrate the skin of the prey.In contrast,C.capillata were mostly equipped with mastigophore-type nematocysts,which are capable of entangling prey and piercing tough chitinous coverings.To compare their protein profiles,the extracts of these two morphologically distinct nematocysts were fractionated using SDS-PAGE.Both jellyfish venom samples exhibited widely distributed molecular weights under reducing conditions,with NnV(Nemopilema nomurai nematocyst venom)appearing to have a greater number of high-molecular-weight proteins(>40 kDa)than CnV(Cyanea capillata nematocyst venom).Most of the protein bands of CnV ranged from～10 to～120 kDa,while the distribution of molecular masses of NnV was even broader,from～10 to～200 kDa.To analyze the cytotoxic potential of CnV and NnV,the effects of various concentrations of CnV(0.6-2μg/ml)and NnV(0.025-0.8 μg/ml)were tested on L929 cells by CCK8 assay.Both CnV and NnV significantly decreased cell viability in a concentration-dependent manner,yielding IC50 values of 1.4 and 0.28 μg/ml,respectively;these IC50 values indicated that NnV was approximately 5 times more cytotoxic than CnV.3.To further explore the putative toxin proteins in the nematocyst venom,total protein from the nematocyst preparations was fractionated using SDS-PAGE and then submitted to in-gel trypsin digestion followed by LC-MS/MS analysis.A total of 189,309 and 267,286 MS/MS spectra were generated in the proteome analyses of CnV and NnV,respectively.Spectra from MS experiments were searched against the C.capillata and N.nomurai reference protein databases described above.After processing,a total of 345 and 329 proteins were identified and annotated,respectively,in CnV and NnV.Among these annotated proteins,structural proteins,enzymes and toxin-like proteins were the most abundant constituents.In order to ensure the completeness of the proteomic analysis and to avoid omitting potential toxin proteins in the CnV and NnV,all in-gel datasets were also searched against the UniProt animal toxin database and the UniProt database of all cnidarian proteins.After filtration,53 and 69 putative toxins were identified as sting-related toxins in CnV and NnV,respectively.These potential toxins belong to common classes of toxins found in other venomous animals,including some cnidarians(Nematostella vectensis,Hydra vulgaris),snakes(Gloydius ussuriensis,Naja annulifera),spiders(Loxosceles intermedia,Lycosa singoriensis),scorpions(Mesobuthus martensii,Lychas mucronatus)and insects.4.Comparative analysis revealed a similar toxin profile in CnV and NnV,in which the toxins could be classified as proteinases,phospholipases,neurotoxins,cysteine-rich secretory proteins(CRISPs),lectins,pore-forming toxins(PFTs),protease inhibitors,ion channel inhibitors,insecticidal components and other toxins.The constituent ratios of the identified toxins in CnV and NnV were calculated separately,and the proportional distribution of these toxin families varied between the two species.Metalloproteinases,proteases and pore-forming toxins were predominant in NnV,representing 27.5%,18.8%and 8.7%of the identified venom proteins,respectively,while phospholipases,neurotoxins and proteases were the top three identified venom proteins in CnV,accounting for 22.6%,17.0%and 11.3%,respectively.Our findings provide comprehensive information on the molecular diversity of toxins from two common blooming and stinging species of jellyfish in China.Furthermore,the results reveal a possible relationship between venom composition and sting consequences,guiding the development of effective treatments for different jellyfish stings.Part ⅡGuided by the multi-omics results,our lab has also committed to isolating and purifying nematocyst venom components and other bioactive substances from jellyfish.However,during the purification process,we found that the venom components of jellyfish were strongly hydrophobic and poorly stable(heat intolerance,sensitive to changes in pH).Thus,only a few toxin monomers have been successfully isolated and identified all over the world.But when we tried to purify toxins from N.nomurai nematocysts,an interesting phenomenon occurred.Crude venoms extracted from nematocysts had high solubility in water.However,when the crude venoms were put in dialysis tubes with molecular weight cut off 10,000 Da and dialyzed against pure water for 24 h,precipitation was produced in the dialysis tubes.The results demonstrated that the removal of small molecular weight substances by dialysis could obviously decrease water solubility of the crude venoms.Besides,if the crude venoms were directly separated by ion exchange or gel filtration chromatography,the first protein elution peak showed a high ultraviolet absorption value,while ultraviolet absorption value of the later elution peak was quite low.The results indicated that most of the venom proteins were eluted simultaneously in the initial stage,leading to the poor purification effect.To sum up,it was speculated that a group of small molecule substances with high water solubility and strong adsorptive force existed in the jellyfish nematocysts and they might play a vital role in enhancing the hydrophilicity and maintaining the stability and activity of venom proteins.The extracts from jellyfish nematocysts were further purified using desalinisation followed by HPLC reversed-phase C18 column and analysed by electron spray ionization mass spectrometry(ESI-MS).As expected,a series of molecules with MW of 516 Da,645 Da,774 Da,903 Da,1032 Da,1161 Da,1290 Da,and 1419 Da were detected.These molecular weights were all multiples of 129 Da(4～11×),indicating that this group of molecules were constituted of structural units with MW of 129 Da.Amino acid sequences analysis showed that glutamic acid(Glu)was the only component in these polymers.The MW of Glu is 147 Da and the MW of glutamate residue is 129 Da.In forming a structure of multiples of MW 129 Da,there are two possibilities—pyroglutamic acid and cyclo-glutamic acid.Given the particularity of glutamic acid,which could be linked by α-or y-amide bond,four possible structures,namely poly(cyclo-α-glutamic acid),poly(cyclo-y-glutamic acid),poly(pyro-α-glutamic acid)and poly(pyro-γ-glutamic acid)might appear.In order to confirm the precise structure,all the four possible molecules composed of 6 glutamic acid residues were synthesized.Finally,the analytical HPLC results showed that the series of small molecules were a group of cyclo-y-poly glutamic acid(cyclo-y-PGA)consisted of 4-11 glutamic acid residues.Biological functional analysis indicated that cyclo-y-PGA could increase the hydrophilicity and stability of jellyfish nematocyst extract.In addition,toxicity test showed that cyclo-y-PGA did not produce any acute toxic reactions on cells and animals.So in the third part,we will apply cyclo-y-PGA as coating material to synthesize cyclo-y-PGA-coated nanomicelles and clarify the advantages of cyclo-y-PGA as a novel bio-coating material.Part ⅢCommon chemotherapeutic drugs have poor water-solubility,chemical instability and short blood circulation time,and lack tumor specificity,thus limiting their therapeutic effects and even causing serious side effects.Fortunately,the fast development of nanoscale transport systems for chemotherapeutic drugs based on carrier materials could help to enhance the targeting capability and stability of the drugs and prolong their release time.In part Ⅱ,we have successfully isolated and identified an entirely new group of small cyclopolypeptides,cyclo-γ-PGA,from the jellyfish nematocysts,which have a large number of free carboxyl groups just like the normal gamma-polyglutamic acid peptide chains and a stabler cyclic peptide backbone.In this part,we firstly developed the dual-responsive nanomicelles-NLS-LA-PpIX,using photosensitizer protoporphyrin(PpIX)and disulfide bond-containing lipoic acid(LA)as the hydrophobic groups and hydrophilic peptide(NLS)as the hydrophilic groups.Furthermore,this study aims to synthesize cyclo-y-PGA-coated nanomicelles loaded doxorubicin-NLS-LA-PpIX-DOX@cyclo-γ-PGA for evaluating the superiority of cyclo-y-PGA as coating material in improving the stability of nanomicelles and enhancing the cellular uptake of micelles through y-glutamyl transpeptidase(GGT)mediated endocytosis pathway,and it is expected to pinpoint the reduction/light dual-responsive properties of these nanomicelles and evaluate their antitumor effects in vitro and in vivo.The main results are as follows:1.The nanomicelles were prepared by solvent co-volatilization method.Malvern Zetasizer results showed that the nanomicelles coated with cyclo-y-PGA had slightly larger particle size but lower PDI.Besides,non-coated micelles had a positive zeta potential which became negative after incubation with cyclo-y-PGA.Transmission electron microscopy(TEM)showed that the micelles had a circular shape and a thin gray cyclo-y-PGA corona was covered on the surface of non-coated micelles after incubating.These results demostrated that cyclo-y-PGA could be successfully coated on the surface of cationic polymer nanomicelles NLS-LA-PpIX-DOX.2.The stability of coating and non-coating micelles was investigated by determining the release profiles of DOX in PBS with 2 M NaCl or 10%FBS.The release rate of DOX from cyclo-y-PGA coating micelles NLS-LA-PpIX-DOX@cyclo-γ-PGA was very slow in buffer with 2 M NaCl or 10%FBS,which was similar to that in PBS.Less than 20%of DOX was released in 24 h.However,the non-coating micelles NLS-LA-PpIX-DOX demonstrated a more rapid release compared with NLS-LA-PpIX-DOX@cyclo-y-PGA,and the cumulative release of DOX significantly increased to more than 40%in buffer over 24 h.These results revealed that buffer with a high ionic concentration or serum could facilitate the degradation of non-coating micelles and the cyclo-y-PGA coating could enhance the stability of the nanomecelles in these solution;similarly,the cyclo-γ-PGA-coated nanomicelles NLS-LA-PpIX-DOX@cyclo-y-PGA showed less hemolysis(less than 5%),indicating that the cyclo-y-PGA coating could reduce the nonspecific interaction with blood components(erythrocytes and serum)and increase the stability of nanomicelles,thus enhancing the stability of the nanomecelles during blood circulation.3.To evaluate the cellular uptake capacity,HCT-116 cells were incubated with DOX,NLS-LA-PpIX-DOX,NLS-LA-PpIX-DOX@y-PGA and NLS-LA-PpIX-DOX@cyclo-γ-PGA under the same conditions for 4 h.The cellular uptake behaviors were compared and analyzed using confocal microscopy and flow cytometry based on the fluorescent property of DOX.The results showed that the fluorescent intensity of NLS-LA-PpIX-DOX@cyclo-γ-PGA in the HCT-116 cells was slightly higher than that of NLS-LA-PpIX-DOX@γ-PGA and significantly higher than NLS-LA-PpIX-DOX and free DOX,indicating that cyclo-γ-PGA coating could facilitate celluar uptake of nanomicelles and the effect was better than y-PGA coating.Furthermore,the cellular uptake of NLS-LA-PpIX-DOX@cyclo-γ-PGA was greatly suppressed by the GGT inhibitor GGsTop(1 μg/ml),and the suppression was GGsTop concentration-dependent.By contrast,the cellular uptake of NLS-LA-PpIX-DOX was not affected by GGsTop.It was demonstrated that GGT-mediated endocytosis was the major mechanism for cellular uptake of cyclo-y-PGA coating micelles.4.The redox-sensitive biodegradable property of DOX loaded micelles was investigated at 37℃ under different conditions of pH 5.0 with 10 mM GSH and pH 5.0 with 10 μM GSH.The reduction-responsive micelles showed a slow sustained release in pH 5.0 with 10 μM GSH and a rapid release upon exposing to pH 5.0 with 10 mM GSH(mimicking tumor intracellular conditions).Notably,DOX was effectively released from NLS-LA-PpIX-DOX,NLS-LA-PpIX-DOX@y-PGA and NLS-LA-PpIX-DOX@cyclo-y-PGA in elevated GSH environments,indicating that these nanomicelles had good reduction response property due to the existence of LA.The singlet oxygen(1O2)production of nanomicelles under laser irradiation(635 nm,5 mW/cm2)was determined using 2’,7’-dichlorofuorescein diacetate(DCFH-DA)probe.The relative ROS level of NLS-LA-PpIX-DOX@cyclo-y-PGA under light illumination retained a higher content as compared to free PpIX,NLS-LA-PpIX-DOX and NLS-LA-PpIX-DOX@y-PGA group,uncovering that cyclo-y-PGA coating could effectively enhance ROS production due to increased cellular uptake of micelles and photosensitizer PpIX.Furthermore,studies on the effects of photosensitizers showed that the in situ generated 1O2 can not only exert its photodynamic therapy(PDT)effect but also disrupt the membranes of endolysosomes and thus facilitate the endosomal escape of internalized nanocarriers(i.e.,photochemical internalization(PCI)).5.The combined chemotherapy and PDT of different micelles were evaluated in in vitro and in vivo experiments.The CCK8 assay showed that cyclo-γ-PGA coating micelles exhibited stronger cytotoxic effects on HCT-116 cells after 635 nm laser irradiation as compared to free DOX,NLS-LA-PpIX-DOX and NLS-LA-PpIX-DOX@γ-PGA groups.In vivo imaging results showed that cyclo-y-PGA coated micelles could effectively reduce the accumulation of DOX in other organs(heart,liver,spleen,lung and kidney)and enhance tumor targeting ability,thereby reducing the side effects of DOX.Finally,in vivo antitumor studies showed that the growth of the tumor was significantly suppressed in 21 d in the NLS-LA-PpIX-DOX@cyclo-y-PGA micelle(with irradiation)group,indicating that a combination of PDT and chemotherapy could effectively improve the tumor inhibition effect.Additionally,there was obvious weight loss in mice administered with free DOX or NLS-LA-PpIX-DOX,possibly resulting from the remarkable side effects of free DOX.On the contrary,no apparent weight change in NLS-LA-PpIX-DOX@cyclo-γ-PGA group with or without light irradiation was observed,indicating there was negligible systemic toxicity produced by cyclo-y-PGA coating micelles.To sum up,the jellyfish nematocysts derived cyclo-y-PGA with a great biocompatibility is promising as a biological coating to improve the stability of nanomicelles and enhance the cellular uptake of micelles through γ-glutamyl transpeptidase mediated endocytosis pathway,providing an alternative avenue to design potential drug carriers for efficient cancer treatment. |
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