Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.     

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.     

Elucidated lipid structures of various human stratum corneum investigated by electron paramagnetic resonance spectroscopy

Background: Electron paramagnetic resonance (EPR) in conjunction with a slow‐tumbling simulation is useful for defining stratum corneum (SC) lipid structure. The objectives of this investigation were to evaluate and elucidate the lipid structure of various human SC as a function of the depth. Methods: The SC from mid‐volar forearms of human volunteers and a cadaver was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Spin probes were used to monitor SC ordering. EPR spectra were analyzed by an EPR slow‐tumbling simulation. Keratin solution from human epidermis was mixed with 5‐doxylstearic acid (5‐DSA) aqueous solution. Results: EPR analyses of 5‐DSA and CHL probes showed immobilized and mobile spectra, respectively. The simulation for 5‐DSA spectra showed differences in ordering values of the SC as a function of depth. EPR of keratin/5‐DSA showed mobile spectral pattern. In addition, EPR of CHL showed a weak and a broad signal. Conclusions: The keratin/5‐DSA results imply that the most likely location of 5‐DSA probe is in SC lipid. CHL probe may not easily permeate the SC. The EPR results along with the simulation analysis provide elucidated SC lipid structure.     

EPR Studies on Film Formation of Colloidal Dispersions, 1. Site Selectivity and Techniques

For the first time, electron paramagnetic resonance (EPR) spectroscopy of spin probes is applied to the characterisation of colloidal polymer dispersions and of films obtained from them. The different domains of the dispersions and films can be selectively addressed by simply adding various, readily available spin probes to the initial dispersions. Evidence for the localisation of these probes is obtained from the comparison of their rotational correlation times or rotational diffusion tensors, from the observation of local order in the case of surfactant probes, and from the variation of fluorine matrix hyperfine couplings in the case of probes in poly(fluoroalkyl acrylates). The introduced methodology forms the basis for a new approach to studies of film formation, film annealing, and film rewetting by spin probe EPR.     

Targeted tumor theranostics using folate-conjugated and camptothecin-loaded acoustic nanodroplets in a mouse xenograft model

In this study, we aimed to validate the feasibility of receptor-targeted tumor theranostics with folate-conjugated (FA) and camptothecin-loaded (CPT) acoustic nanodroplets (NDs) (collectively termed FA-CPT-NDs). The ND formulation was based on lipid-stabilized low-boiling perfluorocarbon that can undergo acoustic droplet vaporization (ADV) under ultrasound (US) exposure. Conjugation of folate enhanced the selective delivery to tumors expressing high levels of folate receptor (FR) under mediation by the enhanced permeability and retention effect. In vitro and in vivo studies were performed using FR-positive KB and FR-negative HT-1080 cell lines and mouse xenograft tumor models. Simultaneous therapy and imaging were conducted with a clinical US imaging system at mechanical indices of up to 1.4 at a center frequency of 10 MHz. The results demonstrated that FA-CPT-NDs selectively attached to KB cells, but not HT-1080 cells. The targeted ADV caused instant and delayed damage via mechanical disruption and chemical toxicity to decrease the viability of KB cells by up to 45%, a much higher decrease than that achieved by the NDs without folate conjugation. The in vivo experiments showed that FR-mediated targeting successfully enhanced the EPR of FA-CPT-NDs in KB tumors mainly on the tumor periphery as indicated by immunofluorescence microscopy and US B-mode imaging. Treatments with FA-CPT-NDs at a CPT dose of 50 μg/kg inhibited the growth of KB tumors for up to six weeks, whereas treatment with NDs lacking folate produced a 4.6-fold increase in tumor volume. For HT-1080 tumors, neither the treatments with FA-CPT-NDs nor those with the NDs lacking folate presented tumor growth inhibition. In summary, FR-targeted tumor theranostics has been successfully implemented with FA-CPT-NDs and a clinical US unit. The ligand-directed and EPR-mediated accumulation provides active and passive targeting capabilities, permitting the antitumor effects of FA-CPT-NDs to be exerted selectively to FR-positive tumors and simultaneously providing targeted US imaging capabilities.     

Novel Access to Propagation Rate Coefficients of Radical Polymerization by the SP–PLP–EPR Method

A novel method for k_p measurement is presented, in which time‐resolved electron paramagnetic resonance detection of radical concentration after applying a single laser pulse (SP?PLP?EPR) is carried out in conjunction with measuring monomer conversion induced by the laser pulse. The method is particularly useful for the study of fully ionized monomers, as will be outlined by experiments on trimethylaminoethyl methacrylate chloride (TMAEMA, 2‐(methacryloyloxy)‐N,N,N‐trimethylethan‐1‐aminium chloride, 20 wt% in aqueous solution) at 60 °C. In addition to k_p, the method provides termination rate coefficient data. The reliability of the so‐obtained rate coefficients is demonstrated by chemically induced experiments and by measuring TMAEMA conversion after single‐pulse initiation via near‐infrared (NIR) spectroscopy.

     

Engineered peptides for the development of actively tumor targeted liposomal carriers of doxorubicin

Chemotherapy is still the treatment of choice for many types of cancer; but its effectiveness is hampered by dose limiting toxicity. Properly designed delivery systems can overcome this shortcoming by reducing the non-specific distribution and toxicity of chemotherapeutics in healthy organs and at the same time increasing drug concentrations at tumor tissue. In this study, we developed stealth liposomal formulations of doxorubicin (DOX) having a novel stable engineered peptide ligand, namely p18-4, that binds specifically to breast cancer cell line MDA-MB-435 on its surface. The coupling of p18-4 to liposomes was carried out through conventional, post insertion and post conjugation techniques and prepared liposomes were characterized for their size and level of peptide modification. The p18-4 decorated liposomal DOX formulations were then evaluated for their cellular uptake as well as cytotoxicity against the human breast cancer MDA-MB-435 cells. In this context, the effect of coupling technique on the uptake and cytotoxicity of p18-4 liposomal DOX in MDA-MB-435 cells was evaluated. The conventional and post conjugation methods of peptide incorporation were found to be more reliable for the preparation of p18-4 decorated liposomes for active DOX targeting to MDA-MB-435 cells. p18-4 decoration of liposomes by these methods did not have a notable effect on the size of prepared liposomes and DOX release, but increased the uptake and cytotoxicity of encapsulated DOX in MDA-MB-435 cells. The results show a potential for p18-4 decorated liposomes prepared by conventional and post conjugation method for tumor targeted delivery of DOX in breast tumor models.     

Interacting surfaces of neurotoxins and acetylcholine receptor

Binding of neurotoxin II Naja naja oxiana derivatives containing one spin label at various positions (Leu 1, Glu 2, Lys 15, Lys 25, Lys 26, His 31, Lys 44 and Lys 46) to purified solubilized acetylcholine receptor protein (AchR) from Torpedo marmorata was studied by EPR techniques. AchR interaction with several dansylated neurotoxin II derivatives was followed by difference fluorescence spectroscopy. A series of neurotoxin II p-azidobenzoyl derivatives were prepared and in three of them modified lysine residues were identified. In combination, spectroscopic data and photolabling implicate a considerable area of the neurotoxin in association with AchR. Rigidity of the neurotoxin II conformation allowed to regard its binding surface as a mould of the AchR corresponding site and to estimate the minimal size of the latter. Conformation of the long-chain neurotoxins and their binding to AchR are briefly discussed basing on the 1H and 19F NMR studies of neurotoxin I Naja naja oxiana, toxin 3 Naja naja siamensis and its acetylated or trifluoroacetylated derivatives, as well as on AchR interaction with the derivatives spin labeled at Lys 27 and His 71     

Skin oxygenation after topical application of liposome-entrapped benzyl nicotinate as measured by EPR oximetry in vivo: Influence of composition and size

New and improved drug delivery systems are the important subject of much scientific research. The development of formulations that increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with healing processes affected by the level of oxygen. We have use EPR oximetry in vivo to compare the influence of liposomal formulations of different size and composition with that of hydrogel with respect to the action of the entrapped benzyl nicotinate (BN). Following the topical application of BN onto the skin of mice, pO₂ increase was measured by low-frequency EPR as a function of time. The effect of BN was evaluated by 3 different parameters: lag-time, time needed for maximum pO₂ increase, and overall effectiveness expressed by the area under the response-time curve. An increase in skin oxygenation was observed after BN application. The results show that the effect of BN incorporated in liposomes is achieved more rapidly than the effect from hydrophilic gel. The composition of the liposomes significantly affects the time at which BN starts to act and, to a lesser extent, the maximum increase of pO₂ in skin and the effectiveness of BN action. However, the size of the liposomes influences both the effectiveness of BN action and the time at which BN starts to act. After repeated application of liposomes, the pO₂ baseline increased and the response of the skin tissue was faster. Our results demonstrate that EPR oximetry is a useful method for evaluating oxygen changes after drug application and for following the time course of their action.