[Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting

High levels of glucagon-like peptide-1 (GLP-1) receptor expression in human insulinomas and gastrinomas provide an attractive target for imaging, therapy, and intraoperative tumor localization, using receptor-avid radioligands. The goal of this study was to establish a tumor model for GLP-1 receptor targeting and to use a newly designed exendin-4-DTPA (DTPA is diethylenetriaminepentaacetic acid) conjugate for GLP-1 receptor targeting. METHODS: Exendin-4 was modified C-terminally with Lys(40)-NH(2), whereby the lysine side chain was conjugated with Ahx-DTPA (Ahx is aminohexanoic acid). The GLP-1 receptor affinity (50% inhibitory concentration [IC(50)] value) of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 as well as the GLP-1 receptor density in tumors and different organs of Rip1Tag2 mice were determined. Rip1Tag2 mice are transgenic mice that develop insulinomas in a well-defined multistage tumorigenesis pathway. This animal model was used for biodistribution studies, pinhole SPECT/MRI, and SPECT/CT. Peptide stability, internalization, and efflux studies were performed in cultured beta-tumor cells established from tumors of Rip1Tag2 mice. RESULTS: The GLP-1 receptor affinity of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 was found to be 2.1 +/- 1.1 nmol/L (mean +/- SEM). Because the GLP-1 receptor density in tumors of Rip1Tag2 mice was very high, a remarkably high tumor uptake of 287 +/- 62 %IA/g (% injected activity per gram tissue) was found 4 h after injection. This resulted in excellent tumor visualization by pinhole SPECT/MRI and SPECT/CT. In accordance with in vitro data, [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 uptake in Rip1Tag2 mice was also found in nonneoplastic tissues such as pancreas and lung. However, lung and pancreas uptake was distinctly lower compared with that of tumors, resulting in a tumor-to-pancreas ratio of 13.6 and in a tumor-to-lung ratio of 4.4 at 4 h after injection. Furthermore, in vitro studies in cultured beta-tumor cells demonstrated a specific internalization of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4, whereas peptide stability studies indicated a high metabolic stability of the radiopeptide in beta-tumor cells and human blood serum. CONCLUSION: The high density of GLP-1 receptors in insulinomas as well as the high specific uptake of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 in the tumor of Rip1Tag2 mice indicate that targeting of GLP-1 receptors in insulinomas may become a useful imaging method to localize insulinomas in patients, either preoperatively or intraoperatively. In addition, Rip1Tag2 transgenic mice represent a suitable animal tumor model for GLP-1 receptor targeting.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Quantum modeling of bioconjugated nanomaterials

In recent years, the bio-conjugated nanostructured materials have emerged as a new class of materials for the bio-sensing and medical diagnostics applications. In spite of their multi-directional applications, interfacing nanomaterials with bio-molecules has been a challenge due to somewhat limited knowledge about the underlying physics and chemistry behind these interactions and also for the complexity of biomolecules. The main objective of this dissertation is to provide such a detailed knowledge on bioconjugated nanomaterials toward their applications in designing the next generation of sensing devices. Specifically, we investigate the changes in the electronic properties of a boron nitride nanotube (BNNT) due to the adsorption of different bio-molecules, ranging from neutral (DNA/RNA nucleobases) to polar (amino acid molecules). BNNT is a typical member of III-V compounds semiconductors with morphology similar to that of carbon nanotubes (CNTs) but with its own distinct properties. More specifically, the natural affinity of BNNTs toward living cells with no apparent toxicity instigates the applications of BNNTs in drug delivery and cell therapy. Our results predict that the adsorption of DNA/RNA nucleobases on BNNTs amounts to different degrees of modulation in the band gap of BNNTs, which can be exploited for distinguishing these nucleobases from each other. Interestingly, for the polar amino acid molecules, the nature of interaction appeared to vary ranging from Coulombic, van der Waals and covalent depending on the polarity of the individual molecules, each with a different binding strength and amount of charge transfer involved in the interaction. The strong binding of amino acid molecules on the BNNTs explains the observed protein wrapping onto BNNTs without any linkers, unlike carbon nanotubes (CNTs). Additionally, the widely varying binding energies corresponding to different amino acid molecules toward BNNTs indicate to the suitability of BNNTs for the biosensing applications, as compared to the metallic CNTs. The calculated I-V characteristics in these bioconjugated nanotubes predict notable changes in the conductivity of BNNTs due to the physisorption of DNA/RNA nucleobases. This is not the case with metallic CNTs whose transport properties remained unaltered in their conjugated systems with the nucleobases. Collectively, the bioconjugated BNNTs are found to be an excellent system for the next generation sensing devices.

Non-invasive measurement of steroids in fish-holding water: important considerations when applying the procedure to behaviour studies

Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the ‘static’ sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release—and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.

Boron nitride nanotubes : synthesis, characterization, functionalization, and potential applications

Boron nitride nanotubes (BNNTs) are structurally similar to carbon nanotubes (CNTs), but exhibit completely different physical and chemical properties. Thus, BNNTs with various interesting properties may be complementary to CNTs and provide an alternative perspective to be useful in different applications. However, synthesis of high quality of BNNTs is still challenging. Hence, the major goals of this research work focus on the fundamental study of synthesis, characterizations, functionalization, and explorations of potential applications. In this work, we have established a new growth vapor trapping (GVT) approach to produce high quality and quantity BNNTs on a Si substrate, by using a conventional tube furnace. This chemical vapor deposition (CVD) approach was conducted at a growth temperature of 1200 °C. As compared to other known approaches, our GVT technique is much simpler in experimental setup and requires relatively lower growth temperatures. The as-grown BNNTs are fully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), Energy Filtered Mapping, Raman spectroscopy, Fourier Transform Infra Red spectroscopy (FTIR), UV-Visible (UV-vis) absorption spectroscopy, etc. Following this success, the growth of BNNTs is now as convenient as growing CNTs and ZnO nanowires. Some important parameters have been identified to produce high-quality BNNTs on Si substrates. Furthermore, we have identified a series of effective catalysts for patterned growth of BNNTs at desirable or pre-defined locations. This catalytic CVD technique is achieved based on our finding that MgO, Ni or Fe are the good catalysts for the growth of BNNTs. The success of patterned growth not only explains the role of catalysts in the formation of BNNTs, this technique will also become technologically important for future device fabrication of BNNTs. Following our success in controlled growth of BNNTs on substrates, we have discovered the superhydrophobic behavior of these partially vertically aligned BNNTs. Since BNNTs are chemically inert, resistive to oxidation up to ~1000°C, and transparent to UV-visible light, our discovery suggests that BNNTs could be useful as self-cleaning, insulating and protective coatings under rigorous chemical and thermal conditions. We have also established various approaches to functionalize BNNTs with polymeric molecules and carbon coatings. First, we showed that BNNTs can be functionalized by mPEG-DSPE (Polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine), a bio-compatible polymer that helps disperse and dissolve BNNTs in water solution. Furthermore, well-dispersed BNNTs in water can be cut from its original length of >10µm to(>20hrs). This success is an essential step to implement BNNTs in biomedical applications. On the other hand, we have also succeeded to functionalize BNNTs with various conjugated polymers. This success enables the dispersion of BNNTs in organic solvents instead of water. Our approaches are useful for applications of BNNTs in high-strength composites. In addition, we have also functionalized BNNTs with carbon decoration. This was performed by introducing methane (CH4) gas into the growth process of BNNT. Graphitic carbon coatings can be deposited on the side wall of BNNTs with thicknesses ranging from 2 to 5 nm. This success can modulate the conductivity of pure BNNTs from insulating to weakly electrically conductive. Finally, efforts were devoted to explore the application of the wide bandgap BNNTs in solar-blind deep UV (DUV) photo-detectors. We found that photoelectric current generated by the DUV light was dominated in the microelectrodes of our devices. The contribution of photocurrent from BNNTs is not significant if there is any. Implication from these preliminary experiments and potential future work are discussed.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Virus purification, detection and removal

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus vaccine recovery can be as low as 30% due to demand of high final titer, virus transduction inhibitors and presence of empty or defective virus capsids as impurities. We have overcome these shortcomings by recovering a high 64% of infectious virus using an aqueous two-phase system. We used high molecular weight polymer and citrate salt to achieve a good yield and eliminated the major contaminant bovine serum albumin. Viruses are also studied for ensuring pure and safe drinking water. Low pressure microfiltrations are continuously being investigated for water filters as they allow high permeate flux and low fouling. Viruses such as PPV are small enough to pass through the microporous membranes. Control of viruses in water is crucial for public health and we have designed an affinity based membrane filter to capture virus. Nanofibers have a high surface to volume ratio providing a highly accessible surface area for virus adsorption. Chitosan an insoluble, biocompatible and biodegradable polymer was used for adsorbing trimer peptide WRW. About 0.2 μmoles of cysteine terminal WRW peptide was conjugated to amine terminal chitosan using maleimide conjugation chemistry. We achieved 90-99% virus removal from water adjusted to a neutral pH. The virus removal from affinity based chitosan was attributed to electrostatic and hydrophobic driven binding effect.

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Epothilone analogues with benzimidazole and quinoline side chains: chemical synthesis, antiproliferative activity, and interactions with tubulin

A series of epothilone B and D analogues bearing isomeric quinoline or functionalized benzimidazole side chains has been prepared by chemical synthesis in a highly convergent manner. All analogues have been found to interact with the tubulin/microtubule system and to inhibit human cancer cell proliferation in vitro, albeit with different potencies (IC(50) values between 1 and 150 nM). The affinity of quinoline-based epothilone B and D analogues for stabilized microtubules clearly depends on the position of the N-atom in the quinoline system, while the induction of tubulin polymerization in vitro appears to be less sensitive to N-positioning. The potent inhibition of human cancer cell growth by epothilone analogues bearing functionalized benzimidazole side chains suggests that these systems might be conjugated with tumor-targeting moieties to form tumor-targeted prodrugs.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by S. pneumoniae strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (plyNCR) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (n = 2) or combinations of nonvaccine types only (n = 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%, P = 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of S. pneumoniae epidemiology under vaccine selection pressure.

Odorless Preparation Of Thioglycosides And Thio Michael Adducts Of Carbohydrate Derivatives

A generalized, odorless, one-pot methodology has been developed for the preparation of 1,2-trans-thioglycosides and thio-Michael addition products of carbohydrate derivatives through triphenyl phosphine mediated cleavage of disulfides and reaction of the thiolate formed in situ with glycosyl bromides and glycosyl conjugated alkenes.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Single-Molecule Conductance of Functionalized Oligoynes: Length Dependence and Junction Evolution

We report a combined experimental and theoretical investigation of the length dependence and anchor group dependence of the electrical conductance of a series of oligoyne molecular wires in single-molecule junctions with gold contacts. Experimentally, we focus on the synthesis and properties of diaryloligoynes with n = 1, 2, and 4 triple bonds and the anchor dihydrobenzo[b]thiophene (BT). For comparison, we also explored the aurophilic anchor group cyano (CN), amino (NH2), thiol (SH), and 4-pyridyl (PY). Scanning tunneling microscopy break junction (STM-BJ) and mechanically controllable break junction (MCBJ) techniques are employed to investigate single-molecule conductance characteristics. The BT moiety is superior as compared to traditional anchoring groups investigated so far. BT-terminated oligoynes display a 100% probability of junction formation and possess conductance values which are the highest of the oligoynes studied and, moreover, are higher than other conjugated molecular wires of similar length. Density functional theory (DFT)-based calculations are reported for oligoynes with n = 1−4 triple bonds. Complete conductance traces and conductance distributions are computed for each family of molecules. The sliding of the anchor groups leads to oscillations in both the electrical conductance and the binding energies of the studied molecular wires. In agreement with experimental results, BT-terminated oligoynes are predicted to have a high electrical conductance. The experimental attenuation constants βH range between 1.7 nm−1 (CN) and 3.2 nm−1 (SH) and show the following trend: βH(CN) < βH(NH2) < βH(BT) < βH(PY) ≈ βH(SH). DFT-based calculations yield lower values, which range between 0.4 nm−1 (CN) and 2.2 nm−1 (PY).