Contrast Agents Based on Human Serum Albumin and Nitroxides for 1H-MRI and Overhauser-Enhanced MRI.

In cancer diagnostics, magnetic resonance imaging (MRI) uses contrast agents to enhance the distinction between the target tissue and background. Several promising approaches have been developed to increase MRI sensitivity, one of which is Overhauser dynamic nuclear polarization (ODNP)-enhanced MRI (OMRI). In this study, a macromolecular construct based on human serum albumin and nitroxyl radicals (HSA-NIT) was developed using a new synthesis method that significantly increased the modification to 21 nitroxide residues per protein. This was confirmed by electron paramagnetic resonance (EPR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry. Gel electrophoresis and circular dichroism showed no significant changes in the structure of HSA-NITs, and no oligomers were formed during modification. The cytotoxicity of HSA-NITs was comparable to that of native albumin. HSA-NITs were evaluated as potential "metal-free" organic radical relaxation-based contrast agents for 1H-MRI and as hyperpolarizing contrast agents for OMRI. Relaxivities (longitudinal and transversal relaxation rates r1 and r2) for HSA-NITs were measured at different magnetic field strengths (1.88, 3, 7, and 14 T). Phantoms were used to demonstrate the potential use of HSA-NIT as a T1- and T2-weighted relaxation-based contrast agent at 3 T and 14 T. The efficacy of 1H Overhauser dynamic nuclear polarization (ODNP) in liquids at an ultralow magnetic field (ULF, B0 = 92 ± 0.8 µT) was investigated for HSA-NIT conjugates. The HSA-NITs themselves did not show ODNP enhancement; however, under the proteolysis conditions simulating cancer tissue, HSA-NIT conjugates were cleaved into lower-molecular-weight (MW) protein fragments that activate ODNP capabilities, resulting in a maximum achievable enhancement |Emax| of 40-50 and a radiofrequency power required to achieve half of Emax, P1/2, of 21-27 W. The HSA-NIT with a higher degree of modification released increased the number of spin probes upon biodegradation, which significantly enhanced the Overhauser effect. Thus, HSA-NITs may represent a new class of MRI relaxation-based contrast agents as well as novel cleavable conjugates for use as hyperpolarizing contrast agents (HCAs) in OMRI.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Self-Penetrating Oligonucleotide Derivatives: Features of Self-Assembly and Interactions with Serum and Intracellular Proteins.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin.

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.

Revealing light-induced structural shifts in G-quadruplex-porphyrin complexes: a pulsed dipolar EPR study.

The binding of G-quadruplex structures (G4s) with photosensitizers is of considerable importance in medicinal chemistry and drug discovery due to their promising potential in photodynamic therapy applications. G4s can experience structural changes as a result of ligand interactions and light exposure. Understanding these modifications is essential to uncover the fundamental biological roles of the complexes and optimize their therapeutic potential. The structural diversity of G4s makes it challenging to study their complexes with ligands, necessitating the use of various complementary methods to fully understand these interactions. In this study, we introduce, for the first time, the application of laser-induced dipolar EPR as a method to characterize G-quadruplex DNA complexes containing photosensitizers and to investigate light-induced structural modifications in these systems. To demonstrate the feasibility of this approach, we studied complexes of the human telomeric G-quadruplex (HTel-22) with cationic 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP4). In addition to showcasing a new methodology, we also aimed to provide insights into the mechanisms underlying photoinduced HTel-22/TMPyP4 structural changes, thereby aiding in the advancement of approaches targeting G4s in photodynamic therapy. EPR revealed G-quadruplex unfolding and dimer formation upon light exposure. Our findings demonstrate the potential of EPR spectroscopy for examining G4 complexes with photosensitizers and contribute to a better understanding of G4s' interactions with ligands under light.

Effects of Bis(imino)acenaphthene (Bian)-Derived Ligands on the Cytotoxicity, DNA Interactions, and Redox Activity of Palladium(II) Bipyridine Complexes.

A series of heteroleptic bipyridine Pd(II) complexes based on 1,2-bis[(2,6-diisopropylphenyl)imino]acenaphthene (dpp-Bian) or 1,2-bis[(2,4,6-trimethylphenyl)imino]acenaphthene (tmp-Bian) were prepared. All complexes were fully characterized by spectrochemical methods, and their crystal structures were confirmed by X-ray diffraction analysis. The 72 h stability of heteroleptic bipyridine Pd(II) complexes with Bian ligands under physiological conditions was investigated using 1H NMR spectroscopy. The anticancer activity of all complexes was assessed in a panel of cancer cell lines in comparison with uncoordinated ligands and clinically used drugs cisplatin and doxorubicin. The ability of the complexes to bind DNA was investigated using several methods, including EtBr replacement assay, density functional theory calculations, circular dichroism spectroscopy, DNA gel electrophoresis, and TUNEL assay. The electrochemical activity of all complexes and the uncoordinated ligands was studied using cyclic voltammetry, and reactive oxygen species production in cancer cells was investigated using confocal microscopy. Heteroleptic bipyridine PdII-Bian complexes were cytotoxic in a low micromolar concentration range and showed some selectivity toward cancer cells in comparison with noncancerous MRC-5 lung fibroblasts.

Long non-coding RNA DINO promotes cisplatin sensitivity in lung adenocarcinoma via the p53-Bax axis.

Background: The damage-induced non-coding (DINO) RNA is a newly identified long non-coding RNA (lncRNA) found in human cells with DNA damage. The treatment of tumors with cisplatin can induce DNA damage; however, whether the lncRNA DINO is involved in the treatment of non-small cell lung cancer (NSCLC) has not yet been elucidated. Methods: The expression of the lncRNA DINO in lung adenocarcinoma cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The lung adenocarcinoma cell line, A549, and derived cisplatin-resistant cell line, A549R, were selected to construct cell models with lncRNA DINO overexpression or interference via lentiviral transfection. After cisplatin treatment, changes in the apoptosis rate were measured. Changes in the p53-Bax axis were detected by qRT-PCR and Western blot. Cycloheximide (CHX) interference demonstrated the stability of p53 with new protein production induced by the lncRNA DINO. The in vivo experiments involved intraperitoneal injection of nude mice with cisplatin after subcutaneous tumor formation, and the tumor diameters and weights were recorded. Immunohistochemistry and hematoxylin and eosin (H&E) staining were performed following tumor removal. Results: We found that the lncRNA DINO was significantly down-regulated in NSCLC. DINO overexpression enhanced the sensitivity of NSCLC cells to cisplatin, while DINO down-regulation decreased the sensitivity of NSCLC cells to cisplatin. Mechanistic investigation indicated that DINO enhanced the stability of p53 and mediated the activation of the p53-Bax signaling axis. Our results also demonstrated that the lncRNA DINO could partially reverse cisplatin resistance induced by silencing the p53-Bax axis, and could inhibit subcutaneous tumorigenesis in nude mice after cisplatin treatment in vivo. Conclusions: The lncRNA DINO regulates the sensitivity of lung adenocarcinoma to cisplatin by stabilizing p53 and activating the p53-Bax axis, and thus, may be a novel therapeutic target to overcome cisplatin resistance.

pH-Responsible Doxorubicin-Loaded Fe3O4@CaCO3 Nanocomposites for Cancer Treatment.

A magnetic nanocomposite (MNC) is an integrated nanoplatform that combines a set of functions of two types of materials. A successful combination can give rise to a completely new material with unique physical, chemical, and biological properties. The magnetic core of MNC provides the possibility of magnetic resonance or magnetic particle imaging, magnetic field-influenced targeted delivery, hyperthermia, and other outstanding applications. Recently, MNC gained attention for external magnetic field-guided specific delivery to cancer tissue. Further, drug loading enhancement, construction stability, and biocompatibility improvement may lead to high progress in the area. Herein, the novel method for nanoscale Fe3O4@CaCO3 composites synthesis was proposed. For the procedure, oleic acid-modified Fe3O4 nanoparticles were coated with porous CaCO3 using an ion coprecipitation technique. PEG-2000, Tween 20, and DMEM cell media was successfully used as a stabilization agent and template for Fe3O4@CaCO3 synthesis. Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) data were used for the Fe3O4@CaCO3 MNC's characterization. To improve the nanocomposite properties, the concentration of the magnetic core was varied, yielding optimal size, polydispersity, and aggregation ability. The resulting Fe3O4@CaCO3 had a size of 135 nm with narrow size distributions, which is suitable for biomedical applications. The stability experiment in various pH, cell media, and fetal bovine serum was also evaluated. The material showed low cytotoxicity and high biocompatibility. An excellent anticancer drug doxorubicin (DOX) loading of up to 1900 µg/mg (DOX/MNC) was demonstrated. The Fe3O4@CaCO3/DOX displayed high stability at neutral pH and efficient acid-responsive drug release. The series of DOX-loaded Fe3O4@CaCO3 MNCs indicated effective inhibition of Hela and MCF-7 cell lines, and the IC 50 values were calculated. Moreover, 1.5 µg of the DOX-loaded Fe3O4@CaCO3 nanocomposite is sufficient to inhibit 50% of Hela cells, which shows a high prospect for cancer treatment. The stability experiments for DOX-loaded Fe3O4@CaCO3 in human serum albumin solution indicated the drug release due to the formation of a protein corona. The presented experiment showed the "pitfalls" of DOX-loaded nanocomposites and provided step-by-step guidance on efficient, smart, anticancer nanoconstruction fabrication. Thus, the Fe3O4@CaCO3 nanoplatform exhibits good performance in the cancer treatment area.

Fluorinated Human Serum Albumin as Potential 19F Magnetic Resonance Imaging Probe.

Fluorinated human serum albumin conjugates were prepared and tested as potential metal-free probes for 19F magnetic resonance imaging (MRI). Each protein molecule was modified by several fluorine-containing compounds via the N-substituted natural acylating reagent homocysteine thiolactone. Albumin conjugates retain the protein's physical and biological properties, such as its 3D dimensional structure, aggregation ability, good solubility, proteolysis efficiency, biocompatibility, and low cytotoxicity. A dual-labeled with cyanine 7 fluorescence dye and fluorine reporter group albumin were synthesized for simultaneous fluorescence imaging and 19F MRI. The preliminary in vitro studies show the prospects of albumin carriers for multimodal imaging.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

Application of EPR to porphyrin-protein agents for photodynamic therapy.

Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.

Human Serum Albumin Labelled with Sterically-Hindered Nitroxides as Potential MRI Contrast Agents.

Four albumin-nitroxide conjugates were prepared and tested as metal-free organic radical contrast agents (ORCAs) for magnetic resonance imaging (MRI). Each human serum albumin (HSA) carrier bears multiple nitroxides conjugated via homocysteine thiolactones. These molecular conjugates retain important physical and biological properties of their HSA component, and the resistance of their nitroxide groups to bioreduction was retained or enhanced. The relaxivities are similar for these four conjugates and are much greater than those of their individual components: the HSA or the small nitroxide molecules. This new family of conjugates has excellent prospects for optimization as ORCAs.

Reversible Dimerization of Human Serum Albumin.

Pulsed Dipolar Spectroscopy (PDS) methods of Electron Paramagnetic Resonance (EPR) were used to detect and characterize reversible non-covalent dimers of Human Serum Albumin (HSA), the most abundant protein in human plasma. The spin labels, MTSL and OX063, were attached to Cys-34 and these chemical modifications of Cys-34 did affect the dimerization of HSA, indicating that other post-translational modifications can modulate dimer formation. At physiologically relevant concentrations, HSA does form weak, non-covalent dimers with a well-defined structure. Dimer formation is readily reversible into monomers. Dimerization is very relevant to the role of HSA in the transport, binding, and other physiological processes.

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins.

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time TM for HSA labeled with OX063 is 6.3 µs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy.

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near-room temperature. This approach is demonstrated using fullerene-nitroxide and fullerene-triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide-labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.

Biotin-decorated anti-cancer nucleotide theranostic conjugate of human serum albumin: Where the seed meets the soil?

Human serum albumin is playing an increasing role as a drug carrier in clinical settings. Biotin molecules are often used as suitable tags in targeted anti-tumor drug delivery systems. We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anti-cancer fluorinated nucleotide conjugated with a biotinylated dual-labeled albumin. Interestingly, in vitro and in vivo study revealed stronger anti-tumor activity of the non-tagged theranostic conjugate than that of the biotin-tagged conjugate, which can be explained by decreased binding of the biotin-tagged conjugate to cellular receptors. Our study sheds light on the importance of site-specific albumin modification for the design of albumin-based drugs with desirable pharmaceutical properties.

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics.

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and 19F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.

Design of protein homocystamides with enhanced tumor uptake properties for (19)F magnetic resonance imaging.

Straightforward and reliable tools for in vivo imaging of tumors can benefit the studies of cancer development, as well as contribute to successful diagnosis and treatment of cancer. (19)F NMR offers an exceptional quantitative way of in vivo imaging of the infused agents because of the lack of (19)F signals from the endogenous molecules in the body. The purpose of this study is to develop molecular probes with appropriate NMR characteristics and the biocompatibility for in vivo applications using (19)F MRI. We have studied the reaction between perfluorotoluene and homocysteine thiolactone resulting in the formation of N-substituted homocysteine thiolactone derivative. It has been shown that the reaction occurs selectively at the para position. This fluorine-labeled homocysteine thiolactone has been employed for the introduction of a perfluorotoluene group as a (19)F-containing tag into human serum albumin. The modified protein has been studied in terms of its ability to aggregate and promote the formation of free radicals. By comparing the properties of N-perfluorotoluene-homocystamide of albumin with N-homocysteinylated albumin, it has been revealed that blocking of the alpha-amino group of the homocysteine residue in the fluorinated albumin conjugate inhibits the dangerous aggregation process, as well as free radical formation. A dual-labeled albumin-based molecular probe for (19)F MRI and fluorescence microscopy has been obtained by functionalizing the protein with both maleimide of a fluorescent dye and a fluorinated thiolactone derivative. The incubation of cells with this conjugate did not reveal any significant reduction in cell viability with respect to the parent albumin. The perfluorotoluene-labeled albumin has been demonstrated to act as a promising agent for in vivo (19)F MRI.

Ligand-directed acid-sensitive amidophosphate 5-trifluoromethyl-2'-deoxyuridine conjugate as a potential theranostic agent.

Herein, we report a novel strategy to engineer an acid-sensitive anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly conjugating the linoleic acid (LA)-modified branched polyethyleneimine with a chemotherapeutic drug trifluorothymidine. Linoleic acid residues were grafted onto 25 kDa polyethyleneimine (PEI) by treating PEI with linoleic acid chloroanhydride. 5-Trifluoromethyl-2'-deoxyuridine (trifluorothymidine, TFT) was introduced into LA-PEI conjugate by phosphorylating the conjugate with amidophosphate of trifluorothymidine 5'-monophosphate (pTFT), which had been activated by its conversion into the N,N-dimethylaminopyridine derivative. The extent of mononucleotide analog incorporation in the polymer was regulated by the ratio of pTFT to the polymer during the synthesis. Samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT conjugates decreased with increasing nucleotide content, as examined using the MTT method. Due to the presence of fluorine atoms, TFT-based conjugates could be detected directly in the animals by (19)F magnetic resonance imaging. In addition, the presence of the amidophosphate group in PEI-LA-pTFT conjugates allowed their detection by in vivo(31)P NMR spectroscopy. Indeed, the (31)P NMR signal of a phosphoramide (δ ~ 12 ppm) was observed in the mouse muscle tissue treated with PEI-LA-pTFT conjugate along with the signals from endogenous phosphorus-containing compounds. At the same time, the use of PEI-LA-pTFT conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugates resulted in prolongation of the animals' life and to the complete disappearance of the tumor after three injections.