Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro.

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over "immortalized" cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (>10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.

Surface functionalization of exosomes using click chemistry.

A method for conjugation of ligands to the surface of exosomes was developed using click chemistry. Copper-catalyzed azide alkyne cycloaddition (click chemistry) is ideal for biocojugation of small molecules and macromolecules to the surface of exosomes, due to fast reaction times, high specificity, and compatibility in aqueous buffers. Exosomes cross-linked with alkyne groups using carbodiimide chemistry were conjugated to a model azide, azide-fluor 545. Conjugation had no effect on the size of exosomes, nor was there any change in the extent of exosome adherence/internalization with recipient cells, suggesting the reaction conditions were mild on exosome structure and function. We further investigated the extent of exosomal protein modification with alkyne groups. Using liposomes with surface alkyne groups of a similar size and concentration to exosomes, we estimated that approximately 1.5 alkyne groups were present for every 150 kDa of exosomal protein.

The role of Akt/GSK-3beta signaling in familial hypertrophic cardiomyopathy.

Mutations in cardiac troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). Transgenic mice expressing a missense mutation (R92Q) or a splice site donor mutation (Trunc) in the cardiac TnT gene have mutation-specific phenotypes but mice of both models have smaller hearts compared to wild type and exhibit hemodynamic dysfunction. Because growth-related signaling pathways in the hearts of mice expressing TnT mutations are not known, we evaluated the impact of increased Akt or glycogen synthase kinase-3beta (GSK-3beta) activity in both mutant TnT mice; molecules that increase heart size via physiologic pathways and block pathologic growth, respectively. Expression of activated Akt dramatically augments heart size in both R92Q and Trunc mice; however, this increase in heart size is not beneficial, since Akt also increases fibrosis in both TnT mutants and causes some pathologic gene expression shifts in the R92Q mice. Activated GSK-3beta results in further decreases in left ventricular size in both R92Q and Trunc hearts, but this decrease is associated with significant mutation-specific phenotypes. Among many pathologic consequences, activating GSK-3beta in R92Q hearts decreases phosphorylation of troponin I and results in early mortality. In contrast, increased GSK-3beta activity in Trunc hearts does not significantly impact cardiac phenotypes. These findings demonstrate that increased Akt and its downstream target, GSK-3beta can impact both cardiac size and phenotype in a mutation-specific manner. Moreover, increased activity of these molecules implicated in beneficial cardiac phenotypes exacerbates the progression of disease in the R92Q TnT mutant.

Biodistribution and delivery efficiency of unmodified tumor-derived exosomes.

The use of exosomes as a drug delivery vehicle has gained considerable interest. To establish if exosomes could be utilized effectively for drug delivery, a better understanding of their in vivo fate must be established. Through comparisons to liposomal formulations, which have been studied extensively for the last thirty years, we were able to make some comprehensive conclusions about the fate of unmodified tumor-derived exosomes in vivo. We observed a comparable rapid clearance and minimal tumor accumulation of intravenously-injected exosomes, PC:Chol liposomes, and liposomes formulated with the lipid extract of exosomes, suggesting that the unique protein and lipid composition of exosomes does not appreciably impact exosomes' rate of clearance and biodistribution. This rapid clearance along with minimal tumor accumulation of unmodified exosomes limits their use as an anti-cancer drug delivery vehicle; however, when delivered intratumorally, exosomes remained associated with tumor tissue to a significantly greater extent than PC:Chol liposomes. Furthermore, experiments utilizing mice with impaired adaptive or innate immune systems, revealed the significance of the innate immune system along with the complement protein C5 on exosomes' rate of clearance.

Application of a ultra performance liquid chromatography method in the determination of DNA quality and stability.

The development of plasmid DNA as a pharmaceutical requires that integrity (i.e., supercoil content) be monitored as part of quality control. The standard method of determining supercoil content is gel electrophoresis followed by staining and imaging, which is complicated by a variety of factors. Previously described chromatographic methods used to quantify supercoil content have had difficulty obtaining reliable separation of the different isoforms. Using ultra performance liquid chromatography, we have optimized buffer conditions, and utilized increased column temperatures in developing a method that allows accurate quantification of each isoform by ultraviolet detection. We found that increasing the column temperature to 55°C improved separation of the isoform peaks as well as increased the resolution of each peak. We demonstrate the utility of this method by quantifying supercoil content of samples subjected to sonication, acidification or lyophilization, and storage. Our results demonstrate that this method allows for a precise quantification of individual DNA isoforms within a heterogeneous sample.

Moisture content impacts the stability of DNA adsorbed onto gold microparticles.

Particle-mediated epidermal delivery (PMED) of small quantities of DNA (0.5-4.0 µg) has been reported to both induce an immune response and protect against disease in human subjects. In order for the PMED of DNA to be a viable technique for vaccination, the adsorbed DNA must be stable during shipping and storage. Here, we report that the storage stability of plasmid DNA adsorbed to 2-µm gold particles is strongly dependent on sample water content. Gold/DNA samples stored at 60°C and 6% relative humidity (RH) maintained supercoil content after 4-month storage, whereas storage at higher RHs facilitated degradation. Storage with desiccants had stabilizing effects on DNA similar to storage at 6% RH. However, storage with "indicating" Drierite and phosphorus pentoxide resulted in enhanced rates of DNA degradation.