Rett syndrome causing missense mutations in the methyl-CpG-binding domain (MBD) of methyl CpG-binding protein 2 (MeCP2) were investigated both in silico and in vitro to reveal their effect on protein stability. It is demonstrated that the vast majority of frequently occurring mutations in the human population indeed alter the MBD folding free energy by a fraction of a kcal/mol up to more than 1 kcal/mol. While the absolute magnitude of the change of the free energy is small, the effect on the MBD functionality may be substantial since the folding free energy of MBD is about 2 kcal/mol only. Thus, it is emphasized that the effect of mutations on protein integrity should be evaluated with respect to the wild-type folding free energy but not with the absolute value of the folding free energy change. Furthermore, it was observed that the magnitude of the effect is correlated neither with the burial of the mutation sites nor with the basic amino acid physicochemical property change. Mutations that strongly perturb the immediate structural features were found to have little effect on folding free energy, while very conservative mutations resulted in large changes of the MBD stability. This observation was attributed to the protein's ability to structurally relax and reorganize to reduce the effect of mutation. Comparison between in silico and in vitro results indicated that some Web servers perform relatively well, while the free energy perturbation approach frequently overpredicts the magnitude of the free energy change especially when a charged amino acid is involved.

Current chemotherapy treatments are limited by poor drug solubility, rapid drug clearance and systemic side effects. Additionally, drug penetration into solid tumors is limited by physical diffusion barriers [e.g., extracellular matrix (ECM)]. Nanoparticle (NP) blood circulation half-life, biodistribution and ability to cross extracellular and cellular barriers will be dictated by NP composition, size, shape and surface functionality. Here, we investigated the effect of surface charge of poly(lactide)-poly(ethylene glycol) NPs on mediating cellular interaction. Polymeric NPs of equal sizes were used that had two different surface functionalities: negatively charged carboxyl (COOH) and neutral charged methoxy (OCH3). Cellular uptake studies showed significantly higher uptake in human brain cancer cells compared to noncancerous human brain cells, and negatively charged COOH NPs were uptaken more than neutral OCH3 NPs in 2D culture. NPs were also able to load and control the release of paclitaxel (PTX) over 19 days. Toxicity studies in U-87 glioblastoma cells showed that PTX-loaded NPs were effective drug delivery vehicles. Effect of surface charge on NP interaction with the ECM was investigated using collagen in a 3D cellular uptake model, as collagen content varies with the type of cancer and the stage of the disease compared to normal tissues. Results demonstrated that NPs can effectively diffuse across an ECM barrier and into cells, but NP mobility is dictated by surface charge. In vivo biodistribution of OCH3 NPs in intracranial tumor xenografts showed that NPs more easily accumulated in tumors with less collagen. These results indicate that a robust understanding of NP interaction with various tumor environments can lead to more effective patient-tailored therapies.