Methylated tetra-amide derivatives of paramagnetic complexes for magnetic resonance biosensing with both BIRDS and CEST.

Paramagnetic agents that utilize two mechanisms to provide physiological information by magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) are described. MRI with chemical exchange saturation transfer (CEST) takes advantage of the agent's exchangeable protons (e.g., -OH or -NHx , where 2 ≥ x ≥ 1) to create pH contrast. The agent's incorporation of non-exchangeable protons (e.g., -CHy , where 3 ≥ y ≥ 1) makes it possible to map tissue temperature and/or pH using an MRSI method called biosensor imaging of redundant deviation in shifts (BIRDS). Hybrid probes based upon 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelate (DOTA4- ) and its methylated analog (1,4,7,10-tetraazacyclododecane-α, α', α″, α‴-tetramethyl-1,4,7,10-tetraacetate, DOTMA4- ) were synthesized, and modified to create new tetra-amide chelates. Addition of several methyl groups per pendent arm of the symmetrical chelates, positioned proximally and distally to thulium ions (Tm3+ ), gave rise to favorable BIRDS properties (i.e., high signal-to-noise ratio (SNR) from non-exchangeable methyl proton peaks) and CEST responsiveness (i.e., from amide exchangeable protons). Structures of the Tm3+ probes elucidate the influence of methyl group placement on sensor performance. An eight-coordinate geometry with high symmetry was observed for the complexes: Tm-L1 was based on DOTA4- , whereas Tm-L2 and Tm-L3 were based on DOTMA4- , where the latter contained an additional carboxylate at the distal end of each arm. The distance of Tm3+ from terminal methyl carbons is a key determinant for sustaining BIRDS temperature sensitivity without compromising CEST pH contrast; however, water solubility was influenced by introduction of hydrophobic methyl groups and hydrophilic carboxylate. Combined BIRDS and CEST detection of Tm-L2, which features two high-SNR methyl peaks and a strong amide CEST peak, should enable simultaneous temperature and pH measurements for high-resolution molecular imaging in vivo.

Imaging Hallmarks of the Tumor Microenvironment in Glioblastoma Progression.

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment.

Comprehensive analysis of liposome formulation parameters and their influence on encapsulation, stability and drug release in glibenclamide liposomes.

An understanding of the factors that affect liposome size, drug loading, stability and drug release is critical for the rational design of liposomes with desired pharmacokinetics and biodistribution. This article presents a report on the formulation and characterization of BIIB093 (glibenclamide) liposomes as well as a detailed analysis of the influence of formulation methods and parameters on encapsulation efficiency, liposome size, charge (zeta potential, ZP), polydispersity index (PDI), and drug release. PEGylated liposomes containing BIIB093 were made using ethanol injection and calcium acetate remote loading. The critical formulation parameters investigated include: the effect of lipid chain length, lipid unsaturation, lipid phase transition temperature (Tc) and the amount of cholesterol. Liposomes generated in this study had low average particle size (130 ± 20 nm), PDI (0.15 ± 0.1) and ZP (-2 ± 1 mV). Liposomes made from lipids with long acyl chains showed enhanced drug loading, encapsulation efficiency and drug retention. Similarly, liposomes made from lipids with high degree of unsaturation and low Tc exhibited faster drug release rates. Additionally, increasing the amount of cholesterol in the liposome bilayer improved PDI, decreased drug incorporation and accelerated drug release but had negligible impact on liposome size and ZP. Furthermore, encapsulating the drug in the liposome core enabled sustained drug release.

Design of Gadoteridol-Loaded Cationic Liposomal Adjuvant CAF01 for MRI of Lung Deposition of Intrapulmonary Administered Particles.

Designing effective and safe tuberculosis (TB) subunit vaccines for inhalation requires identification of appropriate antigens and adjuvants and definition of the specific areas to target in the lungs. Magnetic resonance imaging (MRI) enables high spatial resolution, but real-time anatomical and functional MRI of lungs is challenging. Here, we describe the design of a novel gadoteridol-loaded cationic adjuvant formulation 01 (CAF01) for MRI-guided vaccine delivery of the clinically tested TB subunit vaccine candidate H56/CAF01. Gadoteridol-loaded CAF01 liposomes were engineered by using a quality-by-design approach to (i) increase the mechanistic understanding of formulation factors governing the loading of gadoteridol and (ii) maximize the loading of gadoteridol in CAF01, which was confirmed by cryotransmission electron microscopy. The encapsulation efficiency and loading of gadoteridol were highly dependent on the buffer pH due to strong attractive electrostatic interactions between gadoteridol and the cationic lipid component. Optimal gadoteridol loading of CAF01 liposomes showed good in vivo stability and safety upon intrapulmonary administration into mice while generating 1.5-fold MRI signal enhancement associated with approximately 30% T1 relaxation change. This formulation principle and imaging approach can potentially be used for other mucosal nanoparticle-based formulations, species, and lung pathologies, which can readily be translated for clinical use.

Mapping Extracellular pH of Gliomas in Presence of Superparamagnetic Nanoparticles: Towards Imaging the Distribution of Drug-Containing Nanoparticles and Their Curative Effect on the Tumor Microenvironment.

Since brain's microvasculature is compromised in gliomas, intravenous injection of tumor-targeting nanoparticles containing drugs (D-NPs) and superparamagnetic iron oxide (SPIO-NPs) can deliver high payloads of drugs while allowing MRI to track drug distribution. However, therapeutic effect of D-NPs remains poorly investigated because superparamagnetic fields generated by SPIO-NPs perturb conventional MRI readouts. Because extracellular pH (pHe) is a tumor hallmark, mapping pHe is critical. Brain pHe is measured by biosensor imaging of redundant deviation in shifts (BIRDS) with lanthanide agents, by detecting paramagnetically shifted resonances of nonexchangeable protons on the agent. To test the hypothesis that BIRDS-based pHe readout remains uncompromised by presence of SPIO-NPs, we mapped pHe in glioma-bearing rats before and after SPIO-NPs infusion. While SPIO-NPs accumulation in the tumor enhanced MRI contrast, the pHe inside and outside the MRI-defined tumor boundary remained unchanged after SPIO-NPs infusion, regardless of the tumor type (9L versus RG2) or agent injection method (renal ligation versus coinfusion with probenecid). These results demonstrate that we can simultaneously and noninvasively image the specific location and the healing efficacy of D-NPs, where MRI contrast from SPIO-NPs can track their distribution and BIRDS-based pHe can map their therapeutic impact.

Improved specific loss power on cancer cells by hyperthermia and MRI contrast of hydrophilic Fex Co1-x Fe2 O4 nanoensembles.

Ferrite-based ferri/superparamagnetic nanoparticles can be rapidly heated by an external alternating magnetic field (AMF) to induce tissue necrosis of the adjacent microenvironment, but in addition provide magnetic resonance imaging (MRI) contrast utilizing enhanced water relaxivity. Here we characterized nanoensembles of Fe-Co mixed spinel ferrites (i.e. Fex Co1-x Fe2 O4 , where x ranges from 0.2 to 0.8) synthesized by chemical co-precipitation. With nanoensembles of increasing Co content the saturation magnetization improved, while lattice parameter remained relatively constant. MRI water (transverse) relaxivity at 11.7 T was also boosted with increasing Co content. Efficiency of AMF-induced heating was quite comparable for the nanoensembles with either chitosan or polyethylene glycol (PEG) coating except for PEG-coated Fe0.2 Co0.8 Fe2 O4 , which was twice as less efficient as others. While toxicity of the nanoensembles with either coating examined on 9L tumor cell cultures showed no significant differences, upon AMF exposure (i.e. heat-induced necrosis) Fex Co1-x Fe2 O4 composition with different values of x showed quite dramatic effects on cell death of tumor cells with both coatings. This study lays the ground work for further characterization of other mixed spinel ferrites, and in addition we expect that chitosan and PEG coated Fex Co1-x Fe2 O4 of all the compositions will have good potential for preclinical applications in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Towards longitudinal mapping of extracellular pH in gliomas.

Biosensor imaging of redundant deviation in shifts (BIRDS), an ultrafast chemical shift imaging technique, requires infusion of paramagnetic probes such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis methylene phosphonate (DOTP(8-) ) complexed with thulium (Tm(3+) ) ion (i.e. TmDOTP(5-) ), where the pH-sensitive resonances of hyperfine-shifted non-exchangeable protons contained within the paramagnetic probe are detected. While imaging extracellular pH (pHe ) with BIRDS meets an important cancer research need by mapping the intratumoral-peritumoral pHe gradient, the surgical intervention used to raise the probe's plasma concentration limits longitudinal scans on the same subject. Here we describe using probenecid (i.e. an organic anion transporter inhibitor) to temporarily restrict renal clearance of TmDOTP(5-) , thereby facilitating molecular imaging by BIRDS without surgical intervention. Co-infusion of probenecid with TmDOTP(5-) increased the probe's distribution into various organs, including the brain, compared with infusing TmDOTP(5-) alone. In vivo BIRDS data using the probenecid-TmDOTP(5-) co-infusion method in rats bearing RG2, 9 L, and U87 brain tumors showed intratumoral-peritumoral pHe gradients that were unaffected by the probe dose. This co-infusion method can be used for pHe mapping with BIRDS in preclinical models for tumor characterization and therapeutic monitoring, given the possibility of repeated scans with BIRDS (e.g. over days and even weeks) in the same subject. The longitudinal pHe readout by the probenecid-TmDOTP(5-) co-infusion method for BIRDS adds translational value in tumor assessment and treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of a lanthanide complex encapsulated with MRI contrast agents into liposomes for biosensor imaging of redundant deviation in shifts (BIRDS).

Purposely designed magnetic resonance imaging (MRI) probes encapsulated in liposomes, which alter contrast by their paramagnetic effect on longitudinal (T1) and transverse (T2) relaxation times of tissue water, hold promise for molecular imaging. However, a challenge with liposomal MRI probes that are solely dependent on enhancement of water relaxation is lack of specific molecular readouts, especially in strong paramagnetic environments, thereby reducing the potential for monitoring disease treatment (e.g., cancer) beyond the generated MRI contrast. Previously, it has been shown that molecular imaging with magnetic resonance is also possible by detecting the signal of non-exchangeable protons emanating from paramagnetic lanthanide complexes themselves [e.g., TmDOTP5⁻, which is a Tm³âº -containing biosensor based on a macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate), DOTP5⁻] with a method called biosensor imaging of redundant deviation in shifts (BIRDS). Here, we show that BIRDS is useful for molecular imaging with probes like TmDOTP5⁻ even when they are encapsulated inside liposomes with ultrastrong T1and T2contrast agents (e.g., Magnevist and Molday ION, respectively). We demonstrate that molecular readouts such as pH and temperature determined from probes like TmDOTP5⁻ are resilient, because the sensitivity of the chemical shifts to the probe's environment is not compromised by the presence of other paramagnetic agents contained within the same nanocarrier milieu. Because high liposomal encapsulation efficiency allows for robust MRI contrast and signal amplification for BIRDS, nanoengineered liposomal probes containing both monomers, TmDOTP5⁻ and paramagnetic contrast agents, could allow high spatial resolution imaging of disease diagnosis (with MRI) and status monitoring (with BIRDS).