Targeted cancer therapy: conferring specificity to cytotoxic drugs

The therapeutic activity of most anticancer drugs in clinical use is limited by their general toxicity to proliferating cells, including some normal cells. Although, chemists continue to develop novel cytotoxic agents with unique mechanisms of action, many of these compounds still lack tumor selectivity and have not been therapeutically useful. Monoclonal antibodies that bind to specific markers on the surface of tumor cells offer an alternative therapy that is tumor specific and thus less toxic. Although highly selective, very few monoclonal antibodies are therapeutically useful since they only display modest cell killing activity. The linkage of monoclonal antibodies to highly cytotoxic drugs can be viewed as a means of (a) conferring higher tumor selectivity to cytotoxic drugs that are too toxic to be used on their own or (b) conferring cell killing power to monoclonal antibodies that are tumor-specific but not sufficiently cytotoxic. This Account provides a brief history of the development of antibody-drug conjugates and shows how the lessons learned from the first generation of conjugates has guided the development of more effective antitumor agents. The three components of antibody-drug conjugates, that is, the monoclonal anitbody, the cytotoxic drug, and the linker connecting the drug to the antibody, have been methodically studied and optimized. The antimitotic drug maytansine was chosen for use in the targeted delivery approach because of its high in vitro potency. Analogues of maytansine bearing a disulfide substituent that allowed linkage to monoclonal antibodies via disulfide bonds were prepared. These analogues retain the high potency of the parent drug. The stability of the disulfide link in antibody-maytansinoid conjugates was varied by introduction of methyl substituents on the carbon atoms geminal to the disulfide link. The optimized disulfide linker was stable in circulation in vivo. The circulation half-life of the cytotoxic drug was increased from just a few hours for the unconjugated drug to several days for the conjugate. Upon binding of the conjugate to the tumor cell, internalization and lysosomal processing released the potent cytotoxic agent inside the cell. These conjugates displayed high target-specific cytotoxicity in vitro. The antitumor activity of these targeted agents was superior to that of the antibodies alone or the standard anticancer drugs in human tumor xenograft models. Several conjugates from this new class of tumor-targeted anticancer agents are currrently undergoing clinical evaluation. The progress made in the targeted delivery approach and initial clinical results opens the door to the future development of highly potent drugs that were too toxic on their own to be therapeutically useful.     

A multifunctional ribonuclease A-conjugated carbon dot cluster nanosystem for synchronous cancer imaging and therapy

Carbon dots exhibit great potential in applications such as molecular imaging and in vivo molecular tracking. However, how to enhance fluorescence intensity of carbon dots has become a great challenge. Herein, we report for the first time a new strategy to synthesize fluorescent carbon dots (C-dots) with high quantum yields by using ribonuclease A (RNase A) as a biomolecular templating agent under microwave irradiation. The synthesized RNase A-conjugated carbon dots (RNase A@C-dots) exhibited quantum yields of 24.20%. The fluorescent color of the RNase A@C-dots can easily be adjusted by varying the microwave reaction time and microwave power. Moreover, the emission wavelength and intensity of RNase A@C-dots displayed a marked excitation wavelength-dependent character. As the excitation wavelength alters from 300 to 500 nm, the photoluminescence (PL) peak exhibits gradually redshifts from 450 to 550 nm, and the intensity reaches its maximum at an excitation wavelength of 380 nm. Its Stokes shift is about 80 nm. Notably, the PL intensity is gradually decreasing as the pH increases, almost linearly dependent, and it reaches the maximum at a pH = 2 condition; the emission peaks also show clearly a redshift, which may be caused by the high activity and perfective dispersion of RNase A in a lower pH solution. In high pH solution, RNase A tends to form RNase A warped carbon dot nanoclusters. Cell imaging confirmed that the RNase A@C-dots could enter into the cytoplasm through cell endocytosis. 3D confocal imaging and transmission electron microscopy observation confirmed partial RNase A@C-dots located inside the nucleus. MTT and real-time cell electronic sensing (RT-CES) analysis showed that the RNase A@C-dots could effectively inhibit the growth of MGC-803 cells. Intra-tumor injection test of RNase A@C-dots showed that RNase A@C-dots could be used for imaging in vivo gastric cancer cells. In conclusion, the as-prepared RNase A@C-dots are suitable for simultaneous therapy and in vivo fluorescence imaging of nude mice loaded with gastric cancer or other tumors.     

Vinylhydridosilsesquioxne: Synthesis and characterization of a potential filler material for vinyl-containing polysiloxanes

This paper reports the synthesis and characterization of an organic silsesquioxane (SQ) containing Si vinyl and Si H groups named as vinylhydridosilsesquioxane (SQVH-12). Detailed investigation of the structure of this organosilsesquioxane revealed that SQVH-12 has a highly cross-linked (polycyclic) Si O network of silsesquioxane with Si vinyl and Si H functional groups. Thermogravimetric analysis of the pyrolysis behavior of SQVH-12 yielded a high percentage of ceramic residue (~92%) at 1200°C under an argon atmosphere. The presence of functional groups in SQVH-12 and the high rate of ceramic residue highlights the potential usefulness of SQVH-12 as a filler for vinyl-containing polysiloxanes. Thus, further research of SQVH-12 can find applications in a wide variety of areas such as additive manufacturing, protective coatings, and advanced composites.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

The rnhA gene encoding RNase HI from a psychrotrophic bacterium,Shewanella sp. SIB1, was cloned, sequenced and overexpressedin an rnh mutant strain of Escherichia coli. SIB1 RNase HI iscomposed of 157 amino acid residues and shows 63% amino acidsequence identity to E.coli RNase HI. Upon induction, the recombinantprotein accumulated in the cells in an insoluble form. Thisprotein was solubilized and purified in the presence of 7 Murea and refolded by removing urea. Determination of the enzymaticactivity using M13 DNA–RNA hybrid as a substrate revealedthat the enzymatic properties of SIB1 RNase HI, such as divalentcation requirement, pH optimum and cleavage mode of a substrate,are similar to those of E.coli RNase HI. However, SIB1 RNaseHI was much less stable than E.coli RNase HI and the temperature(T_1/2) at which the enzyme loses half of its activity upon incubationfor 10 min was ~25°C for SIB1 RNase HI and ~60°C forE.coli RNase HI. The optimum temperature for the SIB1 RNaseHI activity was also shifted downward by 20°C compared withthat of E.coli RNase HI. Nevertheless, SIB1 RNase HI was lessactive than E.coli RNase HI even at low temperatures. The specificactivity determined at 10°C was 0.29 units/mg for SIB1 RNaseHI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesisstudies suggest that the amino acid substitution in the middleof the     

Redox and pH dual-responsive biodegradable mesoporous silica nanoparticle as a potential drug carrier for synergistic cancer therapy

Multifunctional mesoporous silica-based nanocarriers able to efficiently encapsulate drugs for stimuli-responsive release and display rapid biodegradation are highly desirable. In this work, we dope disulfide bonds and calcium into silica framework by one step method to obtain a redox and pH dual-responsive biodegradable mesoporous silica nanoparticle (BT-Ca-MSN) as a potential drug carrier for synergistic cancer therapy. TEM and ICP-OES are used to assess the biodegradation behavior of BT-Ca-MSN. The results show that BT-Ca-MSN can significantly biodegrade in a concurrent reductive and acidic environment due to the simultaneous disulfide bonds cleavage and Ca²⁺ release. In addition, BT-Ca-MSN shows efficient drug loading capacity and significant biodegradation-mediated drug release. Moreover, the in-vitro cytotoxicity indicates that BT-Ca-MSN can not only exhibit significant cancer cell killing effect without obvious toxicity on healthy cells via the way of released Ca²⁺-mediated apoptosis, but also can combine with its loaded doxorubicin hydrochloride for synergistic cancer therapy. This work demonstrates that BT-Ca-MSN is a promising platform as drug carrier, providing a paradigm to rationally design biodegradable silica-based carriers for highly efficient cancer therapy.     

Ultrasound-mediated targeted microbubbles: a new vehicle for cancer therapy

With the hope of overcoming the serious side effects, great endeavor has been made in tumor-targeted chemotherapy, and various drug delivery modalities and drug carriers have been made to decrease systemic toxicity caused by chemotherapeutic agents. Scientists from home and abroad focus on the research of targeted microbubbles contrast agent, and the use of the targeted ultrasound microbubble contrast agent can carry gene drugs and so on to the target tissue, as well as mediated tumor cell apoptosis and tumor microvascular thrombosis block, etc., thus plays the role of targeted therapy. Recent studies have elucidated the mechanisms of drug release and absorption, however, much work remains to be done in order to develop a successful and optimal system. In this review, we summarized the continuing efforts in understanding the usage of the ultrasound triggered target microbubbles in cancer therapy, from release mechanism to preparation methods. The latest applications of ultrasound-triggered targeted microbubbles in cancer therapy, especially in gene therapy and antiangiogenic cancer therapy were discussed. Moreover, we concluded that as a new technology, ultrasound–triggered targeted microbubbles used as drug carriers and imaging agents are still energetic and are very likely to be translated into clinic in the near future.     

Retracted: Preparation and characterization of a bacterial cellulose/chitosan composite for potential biomedical application

The following article from the Journal of Applied Polymer Science, “Preparation and Characterization of a Bacterial Cellulose/Chitosan Composite for Potential Biomedical Application” by Cai Zhijiang, Hou Chengwei, and Yang Guang, published online on 3 March 2011 in Wiley OnlineLibrary (J. Appl. Polym. Sci. 2011 , 121, 1488; http://onlinelibrary.wiley.com/doi/10.1002/app.33661/full ), has been retracted by agreement between the authors, the journal's Editor in Chief, Stefano Tonzani, and the publisher, Wiley Periodicals, Inc. The retraction has been agreed due to significant overlap with respect to another article, published in the Journal of Polymer Research ( 2011 , 18, 739) and authored by a subset of the present authors.     

Phosphated crosslinked pectin as a potential excipient for specific drug delivery: preparation and physicochemical characterization

Pectin was chemically modified with different amounts of trisodium trimetaphosphate (STMP) in aqueous solution (pH = 12), thereby giving a material with reduced water solubility. The physiochemical characterization of this new material was carried out through Fourier transform infrared and thermogravimetric analyses. Phosphated pectin (Pect‐STMP) together with prebiotic (oligosaccharide) were incorporated into an aqueous dispersion of polymethacrylate (Eudragit^® RS 30 D) in order to obtain free films using a casting process (50 °C) on a Teflon plate. The free films were evaluated using water vapour transmission, average swelling index in simulated gastric fluid (SGF) and simulated intestinal fluid, scanning electron microscopy and a diffusion study with theophylline in buffer solution with and without pectinolytic enzyme. The results suggest that the new material can be used in the coating process for oral solid‐reservoir systems, to prevent the premature release of drugs in SGF (pH = 1.2). Furthermore, the presence of both Pect‐STMP and oligosaccharide favours the specific degradation of the pellicle by the action of the enzymes produced by colonic microflora. The material obtained in this work has the potential to be applied in devices for drug delivery in the colon, making possible modified release of drugs. Nevertheless, subsequent colon‐specific experiments in vivo need to be carried out in order to confirm the possible application of this new material. Copyright © 2009 Society of Chemical Industry     

Preparation and self-assembly of a dual-functional copolymer for cancer therapy

The pH-responsive amphiphilic copolymer poly(SDMA-co-OEGMA) (PSO) was prepared from the pH-sensitive hydrophobic monomer 2-styryl-1,3-dioxan-5-yl methacrylate (SDMA) and the hydrophilic monomer oligo(ethylene glycol) methyl ether methacrylate (OEGMA) by radical polymerization. Polymeric aggregates with about 130 nm diameter were obtained by the self-assembly of PSO in neutral aqueous solution. The critical aggregation concentration of the copolymer was determined to be 6.5 mg/L (1.2 × 10^?7 M). Cinnamic aldehyde (CA) small molecules are broken away from PSO side chains after the hydrolysis of acid-labile cyclic acetal in cultured A375 human melanoma cells and further suppress the proliferation of this kind of tumor cells. Furthermore, the PSO aggregate was demonstrated to be a drug carrier for encapsulating Nile Red as model drug in in vitro testing. Based on the pH-responsive characteristic, the Nile Red molecules loaded in self-assembly process could be released from the aggregate inside cultured B16 mouse melanoma cells.     

抗人CD4嵌合抗体的稳定表达及鉴定

目的构建抗人CD4嵌合抗体稳定表达细胞株,并对表达产物进行鉴定。方法采用脂质体法将抗人CD4嵌合抗体质粒pHDC4稳定转染CHO-DHFR-细胞,经选择性培养、有限稀释法克隆和MTX加压筛选抗人CD4嵌合抗体稳定表达株,经细胞工厂扩大培养。收集培养上清,经Protein A亲和层析纯化目的抗体,激光共聚焦显微镜观察抗体基因在细胞内的表达,并分别进行质谱分析、蛋白质N-末端测序和平衡解离常数(KD)的测定。结果构建的抗人CD4嵌合抗体稳定表达细胞株的抗体表达水平为4.29～10.52μg/ml;BCA测得纯化回收后的抗体表达水平为12.68 mg/L;激光共聚焦检测显示,抗CD4嵌合抗体稳定表达细胞株可稳定表达人的恒定区和鼠的可变区;质谱分析表明,其含有鼠源性和人源性成分;蛋白质N-末端氨基酸测序表明,其轻链与亲本抗体N-末端氨基酸序列完全一致;其KD为2.67×10-9M。结论成功构建了抗人CD4嵌合抗体稳定表达细胞株,其表达的抗人CD4嵌合抗体保留了亲本抗体的抗原结合特异性和亲和力。     

Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues

Incorporation of the photoisomerizable amino acid phenylazophenylalanine(PAP) into enzyme structures has been proposed as a strategyfor photoswitching enzyme activity. To evaluate the strengthsand limitations of this approach to enzyme photo-control, weperformed a kinetic analysis of RNase S analogues containingPAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containinga single PAP group, the maximum extent of photoconversion (betweenapproximately 96% trans/4% cis and 10% trans/90% cis under standardconditions) sets a limit on the maximum fold change in the initialrate of ~25-fold, if the cis form is the more active isomer,and ~10-fold if the trans form is more active. This extent ofphotoswitching was not realized in the present case becausethe effects of photoisomerization on kinetic constants weresmall and distributed among effects on S-peptide binding, substratebinding and the rate of the chemical step. These results suggestthat photoisomerization could substantially alter enzyme kineticconstants but that a directed combinatorial approach might berequired for realizing maximal photo-control in such systems.The limit set by the extent of photoconversion might be overcomeby coupling multiple PAP groups to one enzyme or by alteringthe behaviour of a system that required oligomerization foractivity.     

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential

The blood clotting enzyme thrombin plays a central role in theaetiology of occlusive disorders such as stroke and acute myocardialinfarction. During fibrinolytic therapy with plasminogen activators,thrombin is neutralized by anticoagulative drugs. In order tocombine plasminogen-activating and thrombin-inhibitory activitieswe constructed chimeric derivatives of recombinant single-chain,urokinase-type plasminogen activator (rscu-PA) which comprisethe kringle and protease domain of rscu-PA fused via a linkersequence to a thrombin-inhibitory domain. The inhibitory domaincontains a sequence element directed to the active site of thrombinand a sequence taken from either hirudin or the human thrombinreceptor both binding to the fibrinogen recognition site ofthrombin. Analysing different sets of point mutants showed thatthe linker between the protease domain and the active sitedirectedsequence is contributing significantly to the thrombin-inhibitorypotential. Kinetic analysis of thrombin inhibition revealedthat most of the chimeras tested competitively inhibit the thrombin-mediatedcleavage of a peptide substrate in a concentration-dependentmanner; however, in two examples the insertion of one glycineresidue into the active site directed-sequence abolished theblockade of the active site. This supports the conclusion thatthe chimeras with high thrombin-inhibitory potential interactwith the active site and the fibrinogen recognition site ofthrombin.     

Preparation and characterization of a Bacterial cellulose/Chitosan composite for potential biomedical application

Bacterial cellulose (BC)/Chitosan (Ch) composite has been successfully prepared by immersing wet BC pellicle in Ch solution followed by freeze-drying process. The morphology of BC/Ch composite was examined by scanning electron microscope (SEM) and compared with pristine BC. SEM images show that Ch molecules can penetrate into BC forming three-dimensional multilayered scaffold. The scaffold has very well interconnected porous network structure and large aspect surface. The composite was also characterized by Fourier transform infrared spectrum, X-ray diffraction, thermogravimetric analysis and tensile test. By incorporation of Ch into BC, crystallinity tends to decrease from 82% to 61%, and the thermal stability increases from 263 °C to 296 °C. At the same time, the mechanical properties of BC/Ch composite are maintained at certain levels between BC and Ch. The biocompatibility of composite was preliminarily evaluated by cell adhesion studies. The cells incubated with BC/Ch scaffolds for 48 h were capable of forming cell adhesion and proliferation. It showed much better biocompatibility than pure BC. Since the prepared BC/Ch scaffolds are bioactive and suitable for cell adhesion, these scaffolds can be used for wound dressing or tissue-engineering scaffolds.     

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

Bovine pancreatic ribonuclease A (RNase A) has been the objectof much landmark work in biological chemistry. Yet the applicationof the techniques of protein engineering to RNase A has beenlimited by problems inherent in the isolation and heterologousexpression of its gene. A cDNA library was prepared from cowpancreas, and from this library the cDNA that codes for RNaseA was isolated. This cDNA was inserted into expression plasmidsthat then directed the production of RNase A in Saccharomycescerevisiae (fused to a modified -factor leader sequence) orEscherichia coli (fused to the pelB signal sequence). RNaseA secreted into the medium by S.cerevisiae was an active buthighly glycosylated enzyme that was recoverable at 1 mg/l ofculture. RNase A produced by E.coli was in an insoluble fractionof the cell lysate. Oxidation of the reduced and denatured proteinproduced active enzyme which was isolated at 50 mg/l of culture.The bacterial expression system is ideal for the large-scaleproduction of mutants of RNase A. This system was used to substitutealanine, asparagine or histidine for Gln11, a conserved residuethat donates a hydrogen bond to the reactive phosphoryl groupof bound substrate. Analysis of the binding and turnover ofnatural and synthetic substrates by the wild-type and mutantenzymes shows that the primary role of Gln11 is to prevent thenon-productive binding of substrate.     

A potential trackable bone filler: Preparation and characterization

This study reported the synthesis of fluorescent hydroxyapatite/alginate/carbon quantum dots (HA/Alg/CQDs) nanocomposites via the co-precipitation technique. The N-doped CQDs as a new class of fluorescent materials were prepared by the citric acid pyrolysis method, with an average size around 4 nm. Physical, chemical, and optical properties of the synthesized nanocomposites were investigated by X-ray diffraction (XRD), Fourier-transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM), field-emission scanning electron microscopy (FESEM), UV–visible spectroscopy, and photoluminescence (PL) spectroscopy, respectively. The PL spectroscopy data verified the favorable in vitro luminescent emission of the HA/Alg/CQDs nanocomposites in comparison with HA/Alg and HA samples. The XRD patterns of the prepared samples confirmed the formation of crystalline HA in all composites, possessing a Ca/P ratio around 1.5 as obtained by EDX elemental analysis. The FESEM analysis exhibited HA nanoplates that homogeneously distributed throughout the alginate matrix. Therefore, the synthesized nanocomposites could be regarded as potential trackable drug carriers for hard tissue engineering applications.     

Expression and characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain flavor ester synthesis

A lipase from Sporisorium reilianum SRZ2 (SRL) with 73% amino acid sequence identity to Candida antarctica lipase B (CALB) was cloned and overexpressed in Pichia pastoris. The recombinant SRL showed a preference for short-chain p-nitrophenyl esters. It achieved maximum activity at pH 8.0 and 65°C for p-nitrophenyl hexanoate (C6) with K_m and k_cat/K_m values of 0.14 mmol∙L⁻¹ and 1712 min⁻¹∙mmol∙L₋₁ at 30°C, respectively. SRL displayed excellent thermostability and pH stability, retaining more than 79% of its initial activity after incubation at 60°C for 72 h and 75% at pH 3 to 11 for 72 h. It also maintained most of its activity in the presence of inhibitors and detergents except sodium dodecyl sulfate, and it tolerated organic solvents. SRL was covalently immobilized and successfully used for ethyl hexanoate synthesis in cyclohexane or in a solvent-free system with a high conversion yield (>95%). Furthermore, high conversion yield was also achieved for the synthesis of various short-chain flavor esters when high substrate concentrations of 2 mol∙L⁻¹ were applied. This study indicated that a CALB-type lipase from S. reilianum SRZ2 showed great potential in organic ester synthesis.     

Preparation and characterization of plasma-sprayed yttria stabilized zirconia as a potential substrate for NOx sensor

Fully stabilized zirconia containing 8 mol% of yttria was synthesized by solid state reaction method and deposited as a thick film by plasma spray coating. The crystal structure and phases developed were characterized by X-ray diffraction (XRD). The surface structure, surface morphology and microstructure developed were studied by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The actual doping content was measured by electron probe micro-analyzer(EPMA). The plasma sprayed YSZ was characterized for its electrolytic properties by ac and dc conductivity measurements. The maximum conductivity for plasma sprayed YSZ was found to be −1.62 Scm⁻¹, which was lesser than the conductivity of standard 8YSZ of −1.03 Scm⁻¹ at pO₂=0.21 atm. However; conductivity trends in the arrhenius plot was observed to be similar for entire YSZ's, suggesting that the conductivity mechanism is same and dominated by oxygen ion conductivity independent of oxygen partial pressure. These promising electrolytic properties of thermal sprayed YSZ suggest that the thermal spray coating method may lead to be used as a potential method for the fabrication of NOx sensor.