Linear-like polypeptide-based micelle with pH-sensitive detachable PEG to deliver dimeric camptothecin for cancer therapy

Nano drug delivery systems have made significant progress in delivering anticancer drugs camptothecin(CPT).However,many challenges for CPT delivery remain,including low drug loading efficiency,premature drug leakage,and poor cellular internalization.Herein,we report a novel dual-sensitive polypeptide-based micelle with remarkably high drug loading of CPT for cancer therapy.This self-assembled micelle possesses the following essential components for CPT:(1)pH-sensitive PEG(OHC-PEG-CHO)for prolonging blood circulation and allowing biocompatibility by shielding the cationic micelles,which can be detached under the tumor acidic microenvironment and facilitates the cellular uptake;(2)polypeptide polylysine-polyphenylalanine(PKF)synthesized via ring-opening polymerization for micelle formation and CPT analogue loading;(3)dimeric CPT(DCPT)with redox-sensitive linker for increasing CPT loading and ensuring drug release at tumor sites.Interestingly,the linear-like morphology of PEG-PKF/DCPT micelles was able to enhance their cellular internalization when compared with the spherical blank PKF micelles.Also,the anticancer efficacy of DCPT against lung cancer cells was significantly improved by the micelle formation.In conclusion,this work provides a promising strategy facilitating the safety and effective application of CPT in cancer therapy.

匀浆提取喜树叶中喜树碱和羟基喜树碱工艺研究

Camptothecine （CPT） and hydroxycamptothecin （HCPT）, two kinds of anti-cancer alkaloids, were extracted from Camptotheca acuminata leaves using homogenate extraction technology under different conditions such as the ratio of material to liquid, ethanol concentration, and homogenate time. The optimum technology parameters for homogenate extraction of CPT and HCPT from C acuminata leaves were determined as homogenate time at 8 rain, ethanol concentration at 55% and the ratio of material to liquid at 1：15 （g：mL）. By using the optimized parameters, we obtained 0.639‰ extraction rate for CPT and 0.437‰ for HCPT. The extraction yields of CPT and HCPT extracted by homogenating technology were higher than those by other extractive methods, such as ultrasonic, reflux, shaking in water bath. It is concluded that the homogenate extraction technology was an efficient method for extracting CPT and HCPT from C acuminata leaves, with characteristics of less extraction time and high yield.

Camptothecin-based nanodrug delivery systems

The drug camptothecin has a wide range of antitumor effects in cancers including gastric cancer,rectal and colon cancer,liver cancer,and lung cancer.Camptothecin-based drugs inhibit topoisomerase 1(Topo 1),leading to destruction of DNA,and are currently being used as important chemotherapeutic agents in clinical antitumor treatment.However,the main obstacle associated with cancer therapy is represented by systemic toxicity of conventional anticancer drugs and their low accumulation at the tumor site.In addition,low bioavailability,poor water solubility,and other shortcomings hinder their anticancer activity.Different from traditional pharmaceutical preparations,nanotechnology-dependent nanopharmaceutical preparations have become one of the main strategies for different countries worldwide to overcome drug development problems.In this review,we summarized the current hotspots and discussed a variety of camptothecin-based nanodrugs for cancer therapy.We hope that through this review,more efficient drug delivery systems could be designed with potential applications in clinical cancer therapy.

The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila

The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry.An example of this is camptothecin,a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants.Recently,camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila.Here,time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O.pumila.It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation.Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels.Accordingly,silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant.Further detailed molecular characterization by electrophoretic mobility shift,yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC.Taken together,the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis.As such,a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

The effects of different nitrogen sources on camptothecin content and related gene expression in Camptotheca acuminata seedlings

Camptotheac acuminata Decne is a unique tree species in China with an important secondary metabolite,camptothecin(CPT),used in the treatment of cancer.Nitrogen(N)is an important element that affects plant growth and the accumulation of CPT.Reports on the effect of N on CPT synthesis from a genetic perspective are scarce.To explore the effects of different N sources and levels on CPT synthesis in C.acuminata,two-year-old seedlings were fertilized with different concentrations of pure ammonium sulphate,source of ammonium N(NH4+-N),and potassium nitrate for nitrate N(NO3--N).Concentrations of 2.5,5,7.5,and10 g pot-1 NH4+-N and NO3--N were used.The results showed that 7.5 g NH4+-N and NO3--N treatments were best for growth and fresh weight of leaves.Compared with the other treatments,the CPT content,tryptophan synthase and tryptophan decarboxylase activities,and expression of the CaTSB and CaTDCl genes under the 2.5 g NH4+-N and NO3--N treatments peaked significantly at 30 days.However,the expression of CaTDC2 surpassed that of the other two genes at 60 days.Therefore,compared with NH4+-N source,the NO3--N source was more beneficial for growth,and NO3--N was better for CPT yield.Consequently,leaves of C.acuminata treated with 2.5 g NO3--N could be harvested after 30 days to obtain maximum CPT content.CaTDC1 is more closely linked to CPT synthesis.The results of this study improved the production of CPT in C.acuminata via fertilization.

HPLC analysis of camptothecin content in various parts of Nothapodytes foetida collected on different periods

Objective:To investigate the content of topoisomerase I-DNA inhibitor alkaloid camptothecin(CPT) from various parts of Nothapodytes foetida(N.foetida) collected from the month of October to February.Methods:The content of CPT was quantified in the methanolic extract of various parts of N.foetida using high performance liquid chromatography(UPLC).Quantification was performed with the regression analysis and the method was validated as per ICH guidelines. Results:The results revealed that maximum concentrations of camptothecin were found in root(2.62%) collected in the month of February followed by fruits(January,1.22%),stem(January,0.81%) and leaves(February,0.70%).Roots were found to have 3-fold higher concentration of CPT than the leaves and stem,while the fruits showed 2-fold higher concentration.Maximum concentration of camptothecin in fruits was observed in month of January,when they were not fully mature, which was 2-fold higher than that of young and fully mature fruits.Conclusions:These findings indicate that the synthesis of CPT differs in different parts of N.foetida and the content varies periodically.

Optimization of ultrasound-assisted extraction of camptothecin from Camptotheca acuminata seeds

Naturally occurring camptothecin（CPT） is an important source of chemotherapeutic agents.The extraction from Camptotheca acuminata is still the main approach to obtain CPT compared with total synthesis.In the present study,ultrasound-assisted extractions（UAE） of CPT from C.acuminata seeds with alkaline solutions were investigated and CPT yield were determined by High Performance Liquid Chromatography.The conditions of alkaline species and concentrations,extraction time,extraction temperature and ultrasonic power were optimized.Results show that both Na3PO4 and Na2CO3 solutions gain good extraction yields,whereas Na3PO4 solution has stronger basicity and need higher concentration than Na2CO3 solution does,thus aqueous Na2CO3 is more beneficial for the extraction.The optimal condition was ultrasonically extracted with 0.5% aqueous Na2CO3 at 50°C and ultrasonic power of 400 W for 60 min.Comparing with UAE with ethanol,the extraction with 0.5% Na2CO3 solution achieves higher yield.Moreover,aqueous Na2CO3 as a solvent has various advantages including non-toxicity,inflammable,non-corrosive and low cost,which ensure this UAE method is a superior method with high utilizing prospect.

20(S)-20-O-Camptothecin β-Aminopropionate: Novel Synthesis and Antitumor Activity in vitro

To improve the biological anticancer activity of 20 （S）-camptothecin, a novel class of 20 （S）-20-O-camptotheein β-aminopropionates were designed and synthesized with eamptothecins as the starting materials, through acylation with acryloyl chloride followed by Michael＇s addition. Twelve esters, 1a-1d, 4a-4d and 5a-5d, were synthesized and evaluated by using MTT assay method. The results demonstrate that all these compounds show a potential eytotoxicity on KB, HT-29, HCT-8, and Bel7402 tumor cell lines. Some compounds, 1d, 4c, 5b, 5c and 5d, show a higher cytoto-xicity on KB and HCT-8 compared with eamptotheein.

Developing new cancer nanomedicines by combination ancient traditional Chinese medicine effective ingredients and advanced nanotechnology

Various effective ingredients extracted from traditional Chinese medicine have achieved more and more attention in the field of cancer therapy due to their high bioactivity and good safety.These traditional Chinese medicine effective ingredients include paclitaxel,arsenical trioxide,camptothecin and so on.However,poor water solubility and low bioavailability of these traditional Chinese medicine ingredients severely hindered their clinical application.The rapid development of nanotechnology provided a new platform,new opportunities and new challenges for their effective delivery in vitro and in vivo.This minireview provides a concentrated summary of the most recent progresses made in this emerging field,highlighting the“combination ancient traditional Chinese medicine ingredients and advanced nanotechnology”strategy for favoring the construction of next generation cancer nanomedicines.

Synthesis and antitumor activity of 9-methyl-10-hydroxycamptothecin

A novel camptothecin analogue, 9-methyl-10-hydroxycamptothecin （4）, was unexpectedly synthesized from 10-hydroxycamptothecin in two steps. The key step included an efficient Mannich-type reaction. The overall yield was 47.2%. An ether analogue of 4, 9-methyl-10-benzylaminomethoxycamptothecin （5）, was also prepared. These new camptothecin analogues were evaluated for in vitro cytotoxicity against four human cancer cell lines, and exhibited more potent antitumor activities than contrals camptothecin and topotecan against several cancer cells.

Synthesis and Insecticidal Activity of Novel Camptothecin Derivatives Containing Analogs of Chrysanthemic Acid Moieties

Creating high-efifcient and environment-friendly pesticides is very important to produce the pollution free agriculture food and maintain the balance of the survival environmental of the human being. According to reports, camptothecin （CPT） and its derivatives are now being explored as a class of botanical insecticide in agriculture due to its novel mode of action. In order to improve the insecticidal activity of CPT, ten novel camptothecin （1） and 10-hydroxycamptothecin （2） derivatives （1a, 1b, 1c, 1d, 1e;2a, 2b, 2c, 2d, 2e） were designed and synthesized via esteriifcation with analogs of chrysanthemic acid, which have outstanding insecticidal activity. The results showed that compound 2a exhibited potent antifeeding effect and the best contact toxicity among the target compounds against the third-instar larvae of beet armyworm, Spodoptera exigua H＆#252;bner. Compound 2a was also found to be the most effective cytotoxic compound to the tested insect cell lines, IOZCAS-Spex-II, which were established from the fat bodies of S. exigua. It was proposed that the 10-hydroxyl group in the camptothecin derivatives is a key factor for the antifeeding activity of a compound. The nature of the substituents was considered the major factor in determining the insecticidal activity of these compounds.

Homogenate extraction technology of camptothecine and hydroxycamptothecin from Camptotheca acuminata leaves

Camptothecine(CPT) and hydroxycamptothecin(HCPT), two kinds of anti-cancer alkaloids, were extracted from Camptotheca acuminata leaves using homogenate extraction technology under different conditions such as the ratio of material to liquid, ethanol concentration, and homogenate time.The optimum technology parameters for homogenate extraction of CPT and HCPT from C.acuminata leaves were determined as homogenate time at 8 min, ethanol concentration at 55% and the ratio of material to liquid at 1:15(g:mL).By using the optimized parameters, we obtained 0.639‰ extraction rate for CPT and 0.437‰ for HCPT.The extraction yields of CPT and HCPT extracted by homogenating technology were higher than those by other extractive methods, such as ultrasonic, reflux, shaking in water bath.It is concluded that the homogenate extraction technology was an efficient method for extracting CPT and HCPT from C.acuminata leaves, with characteristics of less extraction time and high yield.

Preparation,Characterization,and Antitumor Efficacy of Camptothecine-Loaded Hydroxyapatite / Poly( lactic-co-glycolic acid ) Composite Nanofibers via Electrospinning

In the past decade, various medicated nanofibrous scaffolds have been developed as effective drug delivery systems for postsurgical cancer treatment.In this study, hydroxyapatite nanoparticles( HANPs) were used as carriers to load an anticancer agent—camptothecine( CPT),and the CPT-loaded HANPs( CPT@ HANPs) was then incorporated into poly( lactic-co-glycolic acid)( PLGA) nanofibers via electrospinning.Thus fabricated medicated nanofibrous mats( PLGA / CPT @ HANPs) were characterized by field emission scanning electron microscope( FESEM),transmission electron microscope( TEM), attenuated total reflection Fourier transform infrared spectroscopy( ATR-FTIR) and X-ray diffraction( XRD).The release profiles of CPT from the medicated electrospun mats were obtained and their in vitro anticancer efficacy against HeL a cells was also evaluated.The results showed that the CPT was successfully loaded onto the surface of HANPs,and the prepared electrospun mats exhibited a homogeneous and continuous morphology.Furthermore,the loaded CPT exhibited a sustained release behavior from the nanofibrous mats and the released CPT showed a long-term anticancer efficacy against HeL a cells.Therefore,the prepared medicated electrospun mats may be served as an effective drug delivery device for local antitumor treatment.

Sulfuric Acid Catalyzed Preparation of Alkyl and Alkenyl Camptothecin Ester Derivatives and Antitumor Activity against Human Xenografts Grown in Nude Mice

Camptothecin-20-propinate (CZ48) and other camptothecin ester derivatives were prepared by the esterification reac-tions of camptothecin or 9-nitrocamptothecin with the corresponding acylating agents such as organic acid anhydride or chloride with concentrate sulfuric acid as the catalyst. The sulfuric acid-catalyzed reactions gave high yields of camptothecin ester products.Among the 11 compounds prepared by this method, camptothecin-20-O-propionate, camptothecin-20-O-crotonate, and 9-nitrocamptothecin-20-O-propionate showed good anticancer activity against various types of human tumors grown as xenografts in nude mice. The methodology developed for the preparation of camptothecin esters in this article can be applied to a wide scope of other ester derivatives.

OpNAC1 transcription factor regulates the biosynthesis of the anticancer drug camptothecin by targeting loganic acid O-methyltransferase in Ophiorrhiza pumila

Camptothecin(CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis.Two loganic acid O-methyltransferase(OpLAMTs)were identified in strictosidine-producing plant O.pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpL AMT1expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots.Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis.Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

Solvent-mediated handedness inversed and amplified circularly polarized luminescence system based on camptothecin derivative

To realize the handedness controllable circularly polarized luminescence(CPL) system remains challenging. Herein, the solvent-mediated CPL inversion and amplification systems were successfully constructed by camptothecin derivative(CPT-A). Due to the planar structure of N,N-dimethylformamide, it could coassemble with CPT-A, resulting in the alteration of g_(lum) from –0.0082 to +0.0085 by increasing water content. While in the non-planar solvent(hexafluoroisopropanol), the g_(lum) was amplified to 0.034 with the increase in water content. Moreover, the CPT-A could react with the glutathione, resulting in the anticancer drug CPT to make it more toxic to the cancer cells. Overall, the handedness controllable CPL systems were realized by tuning the supramolecular self-assembly of a prodrug.

GSH-responsive camptothecin prodrug-based hybrid micellar nanoparticles enable antitumor chemo-immunotherapy by PD-L1 knockdown

The combinational chemo-immunotherapy as a novel treatment strategy has been widely studied and applied in clinic to enhance antitumor therapeutic efficacy and relieve side effects.RNA interference(RNAi)targeting PD-L1 via inhibiting novo production of PD-L1 will overcome the innate and adaptive PD-L1 expression during chemotherapy,thus enable sustained and efficient immune checkpoint blockade(ICB)to active antitumor immune response.Herein,we designed a glutathione(GSH)-responsive camptothecin(CPT)prodrug-based hybrid micellar nanoparticles(siPD-L1@HM-CPT)to achieve synergistic antitumor chemoimmunotherapy by PD-L1 knockdown.siPD-L1@HM-CPT derived from the one-step loading PD-L1 siRNA(siPD-L1)into the CPT prodrug-based hybrid micelles(HM-CPT)which were co-assembled from biodegradable polyphosphoesters-based prodrug CPT-ss-PAEEP15 and stabilizer DSPE-PEG,showed high loading efficiency,GSH-responsive drug release,and excellent stability and biosafety.siPD-L1@HM-CPT achieved simultaneously the co-delivery of CPT and siPD-L1 in vitro and in vivo,high accumulation at the tumor sites,and rapid intracellular release to promote antitumor efficacy via sensitizing CPT chemotherapy,inducing strong immunogenic cell death(ICD)and sustained ICB to improve intratumoral CD8+T cells infiltration.In addition,the antitumor immunity response limited by the differentiated immunogenicity,intrinsic PD-L1 expression,and intracellular GSH level was facilitated by efficient ICD and ICB from silencing PD-L1 and synergistic CPT chemosensitization in our experimental B16-F10 and 4T1 tumor models.Our study might offer a perspective on designing novel co-delivery nanoparticles by convenient and controllable preparation for antitumor chemo-immunotherapy.

Targeting Tyrosyl-DNA phosphodiesterase I to enhance toxicity of phosphodiester linked DNA-adducts

Our genomic DNA is under constant assault from endogenous and exogenous sources,which needs to be resolved to maintain cellular homeostasis.The eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I(Tdp1)catalyzes the hydrolysis of phosphodiester bonds that covalently link adducts to DNA-ends.Tdp1 utilizes two catalytic histidines to resolve a growing list of DNA-adducts.These DNA-adducts can be divided into two groups:small adducts,including oxidized nucleotides,RNA,and non-canonical nucleoside analogs,and large adducts,such as(drug-stabilized)topoisomerase-DNA covalent complexes or failed Schiff base reactions as occur between PARP1 and DNA.Many Tdp1 substrates are generated by chemotherapeutics linking Tdp1 to cancer drug resistance,making a compelling argument to develop small molecules that target Tdp1 as potential novel therapeutic agents.Tdp1’s unique catalytic cycle,which is centered on the formation of Tdp1-DNA covalent reaction intermediate,allows for two principally different targeting strategies:(1)catalytic inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of DNA-adducts that enhances the effectivity of chemotherapeutics;and(2)poisoning of Tdp1 by stabilization of the Tdp1-DNA covalent reaction intermediate,which would increase the half-life of a potentially toxic DNA-adduct by preventing its resolution,analogous to topoisomerase targeted poisons such as topotecan or etoposide.The catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy best illustrates this concept;however,no small molecules have been reported for this strategy.Herein,we concisely discuss the development of Tdp1 catalytic inhibitors and their results.

喜树碱对B16F10细胞增殖及黑色素合成抑制机理

为研究喜树碱(Camptothecin,CPT)对B16F10小鼠恶性黑色素瘤细胞的增殖及黑色素合成抑制机制,利用噻唑蓝(Methylthiazoletetrazolium,MTT)检测法、显微观察法、Na OH裂解法和多巴氧化法分析不同浓度的CPT对细胞增殖、形态、黑色素合成及酪氨酸酶活性的影响.结果显示:随着CPT浓度的增高和处理时间的延长,B16F10细胞的增殖抑制增强. 160μmol/L CPT处理72 h,B16F10细胞增殖抑制率达77. 0%,一定范围内呈现时间和浓度依赖性(P <0. 05).同时不同浓度CPT对细胞黑色素合成和酪氨酸酶活性也具有明显抑制作用,40μmol/L CPT处理72 h,细胞中黑色素的合成量为85. 37%,酪氨酸酶的活性为56. 4%(P <0. 05).结果表明,喜树碱能有效抑制小鼠黑色素瘤细胞B16F10的增殖和细胞内的黑色素的产生,其机制可能与抑制酪氨酸酶的活性有关.

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: Identification of novel antitumor drug binding site

The studies of novel inhibitors of DNA topoisomerase I (Topo I) have already be-come very promising in cancer chemotherapy. Identifying the new drug-binding residues is playing an important role in the design and optimization of Topo I inhibitors. The designed com-pounds may have novel scaffolds, thus will be helpful to overcome the toxicities of current camptothecin (CPT) drugs and may provide a solution to cross resistance with these drugs. Mul-tiple sequence alignments were performed on eukaryotic DNA topoisomerase I superfamily and thus the evolutionary tree was constructed. The Evolutionary Trace method was applied to iden-tify functionally important residues of human Topo I. It has been demonstrated that class-specific hydrophobic residues Ala351, Met428, Pro431 are located around the 7,9-position of CPT, indi-cating suitable substitution of hydrophobic group on CPT will increase antitumor activity. The conservative residue Lys436 in the superfamily is of particular interest and new CPT derivatives designed based on this residue may greatly increase water solubility of such drugs. It has also been demonstrated that the residues Asn352 and Arg364 were conservative in the superfamily, whose mutation will render CPT resistance. As our molecular docking studies demonstrated they did not make any direct interaction with CPT, they are important drug-binding site residues for future design of novel non-camptothecin lead compounds. This work provided a strong basis for the design and synthesis of novel highly potent CPT derivatives and virtual screening for novel lead compounds.