Functionalized Carbon Nano-Onions as a Smart Drug Delivery System for the Poorly Soluble Drug Carmustine for the Management of Glioblastoma.

The drug delivery system for transporting anticancer agents to targeted tissues in the body is a challenging issue. In search of a suitable biocompatible carrier having controlled and sustained drug release properties of poorly soluble drugs, carbon nano-onions (CNOs) were loaded with an anticancer drug, bis-chloroethyl nitrosourea (BCNU/carmustine). CNOs being autofluorescent, drug-loaded functionalized CNOs (f-CNO-BCNU) can be detected in vivo. Transmission electron microscopy (TEM) and differential light scattering (DLS) techniques were used to analyze the sizes of these f-CNOs. The molecular study revealed that the f-CNO-BCNU readily and noncovalently binds with the folate receptors present on the cancer cell surface in excess. Computer modeling and molecular dynamics simulation followed by binding free energy calculation shows f-CNOs have -29.9 kcal/mol binding free energy, and it noncovalently binds the receptor FRα using loop dynamics of three essential loops present in the protein along with polar stabilization interactions provided by Asp55 and Glu86 residues present in the active site. The f-CNO effectively decreased cancer cell viability with a low IC50 value (the concentration that led to 50% killing of the cells). The cell-based Franz diffusion assay was performed to study the drug release profile. The f-CNO-BCNUs also decreased the mitochondrial membrane potential of U87 cells, increased reactive oxygen species release, and caused a loss of mitochondrial membrane integrity. The f-CNOs also increased the percentage of apoptotic cells observed by the Annexin V assay. Based on observed results, it can be concluded that the f-CNO-BCNU efficiently targets the cancer cells, enhances the bioavailability of carmustine, and can be used as a smart chemotherapeutic agent. This strategy offers better patient compliance and greater bioavailability of the drug.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

A novel strategy to the formulation of carmustine and bioactive nanoparticles co-loaded PLGA biocomposite spheres for targeting drug delivery to glioma treatment and nursing care.

Gliomas are the most common brain tumors in humans. Different chemotherapeutics are available to treat gliomas. However, they are costly and pose numerous side effects. The development of nanocomposite based on chemotherapeutic drug and metallic nanoparticle loaded with polymer could be highly useful against glioma. In this study, carmustine loaded with gold NPs and linked with PLGA-PSPE was produced as a bio-nanocomposite and its efficacy in treating glioma and burn wound were investigated. The synthesized biocomposite was characterized by biophysical techniques. It was observed that the synthesized composite was hexagonal and crystalline nature. TGA analysis showed that the particle had good combustible property. Interestingly, the Cm-Au-PLGA-PSPE composite had exhibited remarkable anti-tumor property against U251 human glioma. The flow cytometry showed a greater increase in the apoptosis rate (62.31%) of glioma cells exposed to the bio-nanocomposite. In addition, a greater reduction in the viability of U251 cells was recorded following treatment with Cm-Au-PLGA-PSPE composite. Quick healing of the heart, liver, spleen, lung and kidney tissue wounds in mouse was noticed with Cm-Au-PLGA-PSPE composite treatment. This study concludes that the newly produced Cm-Au-PLGA-PSPE composite would be a promising alternative in treating human gliomas and associated wounds with increased biomedical applications.

Pep-1&borneol-Bifunctionalized Carmustine-Loaded Micelles Enhance Anti-Glioma Efficacy Through Tumor-Targeting and BBB-Penetrating.

Tumor-targeting and blood-brain barrier (BBB)-penetrating are highly desirable for the treatment of glioma. In this study, we developed Pep-1&borneol-bifunctionalized carmustine-loaded micelles (Pep-1/Bor/CMS-M) capable of targeting interleukin-13 receptor-overexpressed glioma and penetrating the brain microvascular endothelial cells-associated physiologic barriers. Pep-1/Bor/CMS-M were nearly spherical particles with a diameter of 32.6 ± 1.1 nm and zeta potential of -21.3 ± 3.1 mV. Carmustine (CMS) released from Pep-1/Bor/CMS-M in pH 7.4 was significantly faster than in acidic environments. In human glioma BT325 cellular studies, Pep-1/Bor/CMS-M remarkably increased the cytotoxicity, notably improved the internalization, and effectively induced the cell apoptosis. Likewise, in human brain microvascular endothelial cells, Pep-1/Bor/CMS-M obviously promoted the cellular uptake, rapidly decreased the transepithelial electrical resistance, and thereby enhanced the ability of penetration. In orthotopic Luc-BT325 glioma tumor-bearing nude mouse models, the stronger fluorescence signal and longer retention were observed in brain tissues compared with other controls, after single administration of DiD-labeled Pep-1/Bor/M (DiD/Pep-1/Bor/M). Importantly, Pep-1/Bor/CMS-M displayed the strongest inhibition of tumor growth, the longest survival period, and low systemic toxicity in treating orthotopic glioma tumor-bearing nude mice. Simultaneous functionalization of Pep-1 and borneol offers a novel strategy for designing CMS-based nanomedicine and precisely treating glioma.

Inhibition of bcl-2 and cox-2 Protein Expression after Local Application of a New Carmustine-Loaded Clinoptilolite-Based Delivery System in a Chemically Induced Skin Cancer Model in Mice.

Our research has focused on in vitro and in vivo evaluations of a new Carmustine (BCNU)-loaded clinoptilolite-based delivery system. Two clinoptilolite ionic forms-hydrogen form (HCLI) and sodium form (NaCLI)-were prepared, allowing a loading degree of about 5-6 mg BCNU/g of zeolite matrix due to the dual porous feature of clinoptilolite. Clinoptilolite-based delivery systems released 35.23% of the load in 12 h for the BCNU@HCLI system and only 10.82% for the BCNU@NaCLI system. The BCNU@HCLI system was chosen to develop gel and cream semisolid dosage forms. The cream (C_BCNU@HCLI) released 29.6% of the loaded BCNU after 12 h in the Nylon synthetic membrane test and 31.6% in the collagen membrane test, higher by comparison to the gel. The new cream was evaluated in vivo in a chemically induced model of skin cancer in mice. Quantitative immunohistochemistry analysis showed stronger inhibition of B-cell lymphoma-2 (bcl-2) and cyclooxygenase 2 (cox-2) protein expression, known markers for cancer survival and aggressiveness, after the treatment with C_BCNU@HCLI by comparison to all the control treatment types, including an off-label magistral formula commercially available Carmustine cream as reference, bringing evidence that a clinoptilolite-based delivery systems could be used as a cancer drug carriers and controlled release systems (skin-targeted topical delivery systems).

1,3-Bis(2-chloroethyl)-1-nitrosourea-loaded bovine serum albumin nanoparticles with dual magnetic resonance-fluorescence imaging for tracking of chemotherapeutic agents.

To date, knowing how to identify the location of chemotherapeutic agents in the human body after injection is still a challenge. Therefore, it is urgent to develop a drug delivery system with molecular imaging tracking ability to accurately understand the distribution, location, and concentration of a drug in living organisms. In this study, we developed bovine serum albumin (BSA)-based nanoparticles (NPs) with dual magnetic resonance (MR) and fluorescence imaging modalities (fluorescein isothiocyanate [FITC]-BSA-Gd/1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU] NPs) to deliver BCNU for inhibition of brain tumor cells (MBR 261-2). These BSA-based NPs are water dispersible, stable, and biocompatible as confirmed by XTT cell viability assay. In vitro phantoms and in vivo MR and fluorescence imaging experiments show that the developed FITC-BSA-Gd/BCNU NPs enable dual MR and fluorescence imaging for monitoring cellular uptake and distribution in tumors. The T1 relaxivity (R1) of FITC-BSA-Gd/BCNU NPs was 3.25 mM(-1) s(-1), which was similar to that of the commercial T1 contrast agent (R1 =3.36 mM(-1) s(-1)). The results indicate that this multifunctional drug delivery system has potential bioimaging tracking of chemotherapeutic agents ability in vitro and in vivo for cancer therapy.

DNA cleavage at the AP site via ß-elimination mediated by the AP site-binding ligands.

DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase-polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting ß-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase-polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.

AN in vitro evaluation of a carmustine-loaded Nano-co-Plex for potential magnetic-targeted intranasal delivery to the brain.

Targeted delivery of carmustine (BCNU), an efficient brain tumor therapeutic, has been challenged with bioavailability issues due to the Blood Brain Barrier (BBB). The currently effective delivery approach is by implants at the site of the tumor, but this is highly invasive. The intranasal route, which is non-invasive and bypasses the BBB, may be alternative route for delivering BCNU to the brain. In this work, polyvinyl alcohol/polyethyleneimine/fIuorecein isothiocyanate complex (Polyplex) coated iron-oxide nanoparticles (Magnetite) were synthesized employing co-precipitation, epoxidation and EDC/NHS coupling reactions. The Polyplex coated magnetite (Nano-co-Plex) was loaded with BCNU for potential magnetically targeted delivery to the brain following intranasal administration. The Nano-co-Plex was characterized employing Thermogravimetric analysis (TGA), Superconducting Quantum Interference Device (SQUID) magnetometry, Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), X-ray Diffractometry (XRD), Transmission Electron Microscopy (TEM) and Zetasize analysis. Results revealed superparamagnetic hexagonally shaped "core-shell" nanoparticles with cell labeling attributes, of size ranging between 30-50 nm, and a zeta potential value of + 32 ± 2 mV. The Nano-co-Plex synthesized was found to possess high degree of crystallinity with 32% Polyplex coating. The loading and release studies indicated a time-dependent loading with maximum loading capacity of 176.82 µg BCNU/mg of the carrier and maximum release of 75.8% of the loaded BCNU. Cytotoxicity of the BCNU-loaded Nano-co-Plex displayed superiority over the conventional BCNU towards human glioblastoma (HG) cells. Cell studies revealed enhanced uptake and internalization of BCNU-loaded Nano-co-plex in HG cells in the presence of an external magnetic field. These Nano-co-Plexes may be ideal as an intranasal magnetic drug targeting device for BCNU delivery.

Dietary diindolylmethane suppresses inflammation-driven lung squamous cell carcinoma in mice.

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared with mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 µmol/g diet or 2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials.

Honokiol inhibits lung tumorigenesis through inhibition of mitochondrial function.

Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a nonadipogenic PPARγ agonist and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreventive efficacy of honokiol in an initiation model of lung squamous cell carcinoma (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU), which is applied topically, reliably triggering the development of SCC within 24 to 26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol-treated group (P = 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol-treated group. P = 0.004). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1-S cell-cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol changed redox status in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent.

Cationic core-shell nanoparticles with carmustine contained within O6-benzylguanine shell for glioma therapy.

The application of carmustine (BCNU) for glioma treatment is limited due to its poor selectivity for tumor and tumor resistance caused by O6-methylguanine-DNA-methyl transferase (MGMT). To improve the efficacy of BCNU, we constructed chitosan surface-modified poly (lactide-co-glycolides) nanoparticles (PLGA/CS NPs) for targeting glioma, loading BCNU along with O6-benzylguanine (BG), which could directly deplete MGMT. With core-shell structure, PLGA/CS NPs in the diameter around 177 nm showed positive zeta potential. In vitro plasma stability of BCNU in NPs was improved compared with free BCNU. The cellular uptake of NPs increased with surface modification of CS and decreasing particle size. The cytotoxicity of BCNU against glioblastoma cells was enhanced after being encapsulated into NPs; furthermore, with the co-encapsulation of BCNU and BG into NPs, BCNU + BG PLGA/CS NPs showed the strongest inhibiting ability. Compared to free drugs, PLGA/CS NPs could prolong circulation time and enhance accumulation in tumor and brain. Among all treatment groups, F98 glioma-bearing rats treated with BCNU + BG PLGA/CS NPs showed the longest survival time and the smallest tumor size. The studies suggested that the co-encapsulation of BCNU and BG into PLGA/CS NPs could remarkably enhance the efficacy of BCNU, accompanied with greater convenience for therapy.

Microencapsulation of chemotherapeutics into monodisperse and tunable biodegradable polymers via electrified liquid jets: control of size, shape, and drug release.

This paper describes microencapsulation of antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, Carmustine) into biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) using an electrojetting technique. The resulting BCNU-loaded PLGA microcapsules have significantly higher drug encapsulation efficiency, more tunable drug loading capacity, and (3) narrower size distribution than those generated using other encapsulation methods.

Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine.

The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by laromustine with a clinically relevant IC(50) value of 4.65 µM. A derivative of laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for laromustine.

Nimustine hydrochloride: the first crystal structure determination of a 2-chloroethyl-N-nitrosourea hydrochloride derivative by X-ray powder diffraction and solid-state NMR.

Nimustine hydrochloride [systematic name: 4-amino-5-({[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino}methyl)-2-methylpyrimidin-1-ium chloride], C(9)H(14)ClN(6)O(2)(+)·Cl(-), is a prodrug of CENU (chloroethylnitrosourea) and is used as a cytostatic agent in cancer therapy. Its crystal structure was determined from laboratory X-ray powder diffraction data. The protonation at an N atom of the pyrimidine ring was established by solid-state NMR spectroscopy.

The effectiveness of a magnetic nanoparticle-based delivery system for BCNU in the treatment of gliomas.

This study describes the creation and characterization of drug carriers prepared using the polymer poly[aniline-co-N-(1-one-butyric acid) aniline] (SPAnH) coated on Fe(3)O(4) cores to form three types of magnetic nanoparticles (MNPs); these particles were used to enhance the therapeutic capacity and improve the thermal stability of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a compound used to treat brain tumors. The average hydrodynamic diameter of the MNPs was 89.2 ± 8.5 nm and all the MNPs displayed superparamagnetic properties. A maximum effective dose of 379.34 µg BCNU could be immobilized on 1 mg of MNP-3 (bound-BCNU-3). Bound-BCNU-3 was more stable than free-BCNU when stored at 4 °C, 25 °C or 37 °C. Bound-BCNU-3 could be concentrated at targeted sites in vitro and in vivo using an externally applied magnet. When applied to brain tumors, magnetic targeting increased the concentration and retention of bound-BCNU-3. This drug delivery system promises to provide more effective tumor treatment using lower therapeutic doses and potentially reducing the side effects of chemotherapy.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders.

Chemotherapeutic drug transport to brain tumor.

Implantation of polymeric wafers to deliver a chemotherapeutic drug is the most popular strategy against a brain tumor, but the understanding on local drug transport to influence the treatment efficacy is often overlooked. In this work, we employ a computational fluid dynamics simulation to study the suitability of four chemotherapeutic agents from a transport perspective, which specifically are carmustine, paclitaxel, 5-fluorouracil (5-FU), and methotrexate (MTX). The study is based on the diffusion/reaction/convection model, in which Darcy's law is used to account the convective contribution of the interstitial fluid. A realistic three-dimensional (3D) tissue geometry is extracted from magnetic resonance images (MRI) of a brain tumor. Our analysis explains how the distribution of the drug in the brain tumor is sensitively coupled to its physico-chemical properties. For the postulated conditions, only paclitaxel exhibits minimal degradation within the cavity: its effective cavity concentration is at least two times higher than those of others. It also exhibits the best penetration of the remnant tumor, so that the tumor is exposed to higher effective concentration up to two orders of magnitude as compared to others. It is also found that tumor receives uneven distribution of drug concentration, in which, even paclitaxel fails to provide adequate penetration on that part of the cavity surface nearest to the ventricles. In addition, we consider antiangiogenic treatment, which has been postulated to be a way to avoid drug loss from the treatment region by convection. It is shown that convection is of only marginal importance and that renormalization has little effect.

Controlled-release implant system formulated using biodegradable hemostatic gauze as scaffold.

A unique polymer-based sustained-release implant was formulated using biodegradable hemostatic gauze as the scaffold. A piece of commercial gauze, Surgicel was coated with a poly(lactic-co-glycolic) acid (PLGA) solution in which drugs were loaded, followed by evaporating the solvent. Drug release kinetics from the PLGA coating was examined using phenol red (PhR, a hydrophilic dye) and carmustine (BCNU, a hydrophobic anti-tumor agent) as model drugs. With an additional drug-free PLGA over-layer coated on top of the drug-loaded PLGA coating, nearly zero order release was archived for both of the model drugs for a period of 12-14 days when the implants were incubated in PBS buffer at 37 degrees C. However, the drug release rate was independent of types of PLGA polymers such as lactide/glycolide ratio. A degradation study showed that the hydrophilic Surgicel scaffold itself degraded in 3 days of the release incubation regardless of the thickness of the polymer coating on top of it, suggesting that the loaded drug may be released through the diffusion channels created by the scaffold degradation. Characterization of this formulation using XRD and DSC indicated that the drug, BCNU, was distributed in the PLGA matrix in amorphous state. Images of scanning electron microscope showed that PLGA was coated on the outer and inner surfaces of the porous Surgicel.

Synthesis and characterization of DNA containing an N1-2'-deoxyinosine-ethyl-N3-thymidine interstrand cross-link: a structural mimic of the cross-link formed by 1,3-bis-(2-chloroethyl)-1-nitrosourea.

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.