Drug delivery, biodistribution and anti-EGFR activity: theragnostic nanoparticles for simultaneous in vivo delivery of tyrosine kinase inhibitors and kinase activity biosensors

In vivo delivery of small molecule therapeutics to cancer cells, assessment of the selectivity of administration, and measuring the efficacity of the drug in question at the molecule level, are important ongoing challenges in developing new classes of cancer chemotherapeutics. One approach that has the potential to provide targeted delivery, tracking of biodistribution and readout of efficacy, is to use multimodal theragnostic nanoparticles to deliver the small molecule therapeutic. In this paper, we report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes. These simultaneously deliver an inhibitor of the EGFR tyrosine kinase, and plasmid DNA coding for a Crk-based biosensor, Picchu-X, which when expressed in the target cells can be used to quantify the inhibition of EGFR in vivo in a mouse colorectal cancer xenograft model. Reversible bioconjugation of a known analogue of the tyrosine kinase inhibitor Mo-IPQA to a cationic peptide, and co-formulation with peptides containing both EGFR-binding and cationic sequences, allowed for good levels of inhibitor encapsulation with targeted delivery to LIM1215 colon cancer cells. Furthermore, high levels of expression of the Picchu-X biosensor in the LIM1215 cells in vivo allowed us to demonstrate, using fluorescence lifetime microscopy (FLIM)-based biosensing, that EGFR activity can be successfully suppressed by the tyrosine kinase inhibitor, released from the lipopolyplexes. Finally, we measured the biodistribution of lipopolyplexes containing 125I-labelled inhibitors and were able to demonstrate that the lipopolyplexes gave significantly higher drug delivery to the tumors compared with free drug

Effect of Liposomal Encapsulation on the Chemical Exchange Properties of Diamagnetic CEST Agents

Exogenous chemical exchange saturation transfer (CEST) contrast agents such as glucose or 2-deoxy-D-glucose (2-DG) have shown high sensitivities and significant potential for monitoring glucose uptake in tumors with MRI. Here, we show that liposome encapsulation of such agents can be exploited to enhance the CEST signal by reducing the overall apparent exchange rate. We have developed a concise analytical model to describe the liposomal contrast dependence on several parameters such as pH, temperature, irradiation amplitude, and intraliposomal water content. This is the first study in which a model has been constructed to measure the exchange properties of diamagnetic CEST agents encapsulated inside liposomes. Experimentally measured exchange rates of glucose and 2-DG in the liposomal system were found to be reduced due to the intermembrane exchange between the intra- and extraliposomal compartments because of restrictions in water transfer imposed by the lipid membrane. These new theoretical and experimental findings will benefit applications of diamagnetic liposomes to image biological processes. In addition, combining this analytical model with measurements of the CEST signal enhancement using liposomes as a model membrane system is an important new general technique for studying membrane permeabilit

Multi-modal imaging probe for assessing the efficiency of stem cell delivery to orthotopic breast tumours

Stem cells have been utilised as anti-cancer agents due to their ability to home to and integrate within tumours. Methods to augment stem cell homing to tumours are being investigated with the goal of enhancing treatment efficacy. However, it is currently not possible to evaluate both cell localisation and cell viability after engraftment, hindering optimisation of therapy. In this study, luciferase-expressing human adipocyte-derived stem cells (ADSCs) were incubated with Indium-111 radiolabelled iron oxide nanoparticles to produce cells with tri-modal imaging capabilities. ADSCs were administered intravenously (IV) or intracardially (IC) to mice bearing orthotopic breast tumours. Cell fate was monitored using bioluminescence imaging (BLI) as a measure of cell viability, magnetic resonance imaging (MRI) for cell localisation and single photon emission computer tomography (SPECT) for cell quantification. Serial monitoring with multi-modal imaging showed the presence of viable ADSCs within tumours as early as 1-hour post IC injection and the percentage of ADSCs within tumours to be 2-fold higher after IC than IV. Finally, histological analysis was used to validate engraftment of ADSC within tumour tissue. These findings demonstrate that multi-modal imaging can be used to evaluate the efficiency of stem cell delivery to tumours and that IC cell administration is more effective for tumour targeting

Tunable Semiconducting Polymer Nanoparticles with INDT-Based Conjugated Polymers for Photoacoustic Molecular Imaging.

Photoacoustic imaging combines both excellent spatial resolution with high contrast and specificity, without the need for patients to be exposed to ionizing radiation. This makes it ideal for the study of physiological changes occurring during tumorigenesis and cardiovascular disease. In order to fully exploit the potential of this technique, new exogenous contrast agents with strong absorbance in the near-infrared range, good stability and biocompatibility, are required. In this paper, we report the formulation and characterization of a novel series of endogenous contrast agents for photoacoustic imaging in vivo. These contrast agents are based on a recently reported series of indigoid π-conjugated organic semiconductors, coformulated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, to give semiconducting polymer nanoparticles of about 150 nm diameter. These nanoparticles exhibited excellent absorption in the near-infrared region, with good photoacoustic signal generation efficiencies, high photostability, and extinction coefficients of up to three times higher than those previously reported. The absorption maximum is conveniently located in the spectral region of low absorption of chromophores within human tissue. Using the most promising semiconducting polymer nanoparticle, we have demonstrated wavelength-dependent differential contrast between vasculature and the nanoparticles, which can be used to unambiguously discriminate the presence of the contrast agent in vivo

Simultaneous confidence regions for multivariate bioequivalence

Demonstrating bioequivalence of several pharmacokinetic (PK) parameters, such as AUC and Cmax, that are calculated from the same biological sample measurements is in fact a multivariate problem, even though this is neglected by most practitioners and regulatory bodies, who typically settle for separate univariate analyses. We believe, however, that a truly multivariate evaluation of all PK measures simultaneously is clearly more adequate. In this paper, we review methods to construct joint confidence regions around multivariate normal means and investigate their usefulness in simultaneous bioequivalence problems via simulation. Some of them work well for idealised scenarios but break down when faced with real-data challenges such as unknown variance and correlation among the PK parameters. We study the shapes of the confidence regions resulting from different methods, discuss how marginal simultaneous confidence intervals for the individual PK measures can be derived, and illustrate the application to data from a trial on ticlopidine hydrochloride. An R package is available