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.

Camptothecin and 10-Hydroxycamptothecin Accumulate Differentially Under Specific Developmental Control in Camptotheca acuminata

Camptotheca acuminata Decaisne is a tree unique to China, producing terpenoid indole alkaloid (TIA), camptothecin (CPT), and 10-hydroxycamptothecin (HCPT), which possesses strong anti-cancer bioactivity. The dynamics of HCPT and CPT during seed maturation, seed germination, postgerminative development and daily growth were measured by High Performance Liquid Chromatography (HPLC). This article provides some evidence for presenting the theory that each characteristic of HCPT and CPT accumulation is under developmental regulation and then attempt to elucidate the metabolic relationships between them. HCPT accumulates particularly in young tissues and organs and is relatively unstable, while the distribution of CPT is more ubiquitous and stable. Their different distribution properties suggest that HCPT and CPT are differentially regulated and play distinct roles:during development.

Different Responses of Camptothecin and 10-Hydroxycamptothecin to Heat Shock in Camptotheca acuminata Seedlings

Plants interact with their environment by producing a diverse array of secondary metabolites, one of which is alkaloid. In this study, alkaloids, including camptothecin (CPT) and 10-hydroxycamptothecin (HCPT), malondialdehyde (MDA) and chlorophyll contents were measured during heat shock in seedlings of Camptotheca acuminata Decaisne unique to China. Responses of different tissues, including young leaves, old leaves, buds and barks, to heat shock were examined in alkaloid changes. CPT and HCPT concentrations reached their peak values separately at 38 degreesC and 40 degreesC, which were below the lethal heat-shock temperature indicated by MDA and chlorophyll, and their great changes took place in young leaves. These results indicated that CPT and HCPT were involved in the C acuminata resistance against heat shock from its environment. Furthermore, plant rigidly observed the cost-benefit principle and mobilized and allocated limited alkaloid sources to young and reproductive tissues preferentially. In addition, HCPT displayed well-regulated changes during incubation at sublethal temperature, and this indicated that HCPT might play a more positively defensive role in enhancement of plant thermotolerance than CPT does.

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 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.

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.

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.

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.

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.

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.

Effect of planting density on plant growth and camptothecin content of Camptotheca acuminata seedlings

C. acuminata seedlings cultivated in greenhouse were transplanted into the fields with 5 designed planting densities (11, 16, 25,44 and 100 plants·m^-2) in May of 2004 and were harvested in the middle of September of 2004. The seedling growth indexes including plant height and crown width, biomass allocation, camptothecin (CPT) content and CPT yield of different organs (young leaf, old leaf, stem,and root) were studied. For the 5 selected planting densities, the plant biomass, height, crown width, and total leaf area of C. acuminata seedlings all showed highest values at the planting density of 25 plants ·m^-2. CPT content in young leaves was higher than that in other organs of seedlings and presented an obvious change with the variation of planting densities and with the highest value at density of 100plants·m-2, while for other organs no significant variation in CPT content was found with change of planting density. The accumulation of CPT was enhanced significantly at the planting density of 25 plants·m^-2. It is concluded that for the purpose to get raw materials with more CPT from C. acuminata, the optimal planting density of C. acuminata seedlings should be designed as 25 plants·m^-2.

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.

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.

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.

Facile synthesis and cytotoxicity of 5-C-alkylates of 20(S)-camptothecins

A series of 5-C-alkylating camptothecins have been synthesized by one-step method, and their in vitro antitumor activity was evaluated against six human cancer cell lines. The results showed that all 5-C-alkylates of camptothecins possessed poor cytotoxicity on six human cancer cell lines.

Synthesis and cytotoxic activity of 7-alkynyl camptothecin derivatives

Several 7-alkynyl camptothecin derivatives were prepared via Sonogashira coupling. And anti-tumor activities of these compounds were evaluated against human esophageal cancer cell line （Eca-109）, human chronic myeloid leukaemia cell line （K562）, bladder cancer cell line （5637） and gastric cell line （SGC7901）. Compounds 9a--d and 10a exhibited remarkable in vitro cytotoxic activity, compared with topotecan.

Density function theory study on the interaction between camptothecin and cytosine

Density function M06 method has been used to optimize the geometries of camptothecin-cytosine at 6-3 I＋G＊ basis. Finally, thirteen stabilized complexes have been obtained. Theories of atoms in molecules （AIM） and natural bond orbital （NBO） have been utilized to investigate the hydrogen bonds involved in all the complexes. The interaction energies of all the complexes are corrected by basis set superposition error （BSSE）. By the analysis of complexes interaction energy, charge density, second- order interaction energies E（2）; it is indicated that the complex 6 is the most stable structure.

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.

Synthesis and antitumor activity of heterocyclic acid ester derivatives of 20S-camptothecins

A series of heterocyclic acid esters of camptothecins as new compounds had been synthesized by acylation method, their in vitro and in vivo antitumor activities were evaluated. The cytotoxic results showed that 6 possessed the best efficacy on six human cancer cell lines in the six classes of CPTs＇ derivatives. In vivo testing results indicated that 9 had better antitumor activity against mouse liver carcinoma H22 than topotecan.

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.