Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes selfassembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4^+ cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction.All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4^+ human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker.     

MUC1 aptamer-conjugated mesoporous silica nanoparticles effectively target breast cancer cells

In the present study, we developed aptamer (Apt) conjugated mesoporous silica nanoparticles (MSNs) for specific delivery of epirubicin (EPI) to breast cancer cells. MSNs were synthesized and functionalized with 3-mercaptopropyltrimethoxysilane (3-MPTMS), followed by MUC1 aptamer conjugation through disulfide bonds. The nanoparticles were analyzed by transmission electron microscopy (TEM), particle size analyzer, zeta potential, elemental analysis (CHNS), aptamer conjugation efficiency, drug loading efficiency, and drug release profile. Cell uptake and in vitro cytotoxicity of different formulations were performed. The results of MSNs characterization confirmed spherical nanoparticles with thiol functional groups. Particle size of obtained nanoparticles was 163?nm in deionized water. After conjugation of MUC1 aptamer and EPI loading (MSN-MUC1-EPI), particle size increased to 258?nm. The aptamer conjugation to MSNs with disulfide bonds were confirmed using gel retardation assay. Cellular uptake studies revealed better cell uptake of MSN-MUC1-EPI compared to MSN-EPI. Moreover, cytotoxicity study results in MCF7 cell lines showed improved cytotoxicity of MSN-MUC1-EPI in comparison with MSN-EPI or EPI at the same concentration of drug. These results exhibited that MSN-MUC1-EPI has the potential for targeted drug delivery into MUC1 positive breast cancer cells to improve drug efficacy and alleviate side effects.     

Preparation of emulsifying wax/glyceryl monooleate nanoparticles and evaluation as a delivery system for repurposing simvastatin in bone regeneration

Simvastatin (Sim) is a widely known drug in the treatment of hyperlipidemia, which has attracted so much attention in bone regeneration due to its potential osteoanabolic effect. However, repurposing of Sim in bone regeneration will require suitable delivery systems that can negate undesirable off-target/side effects. In this study, we have investigated a new lipid nanoparticle (NP) platform that was fabricated using a binary blend of emulsifying wax (Ewax) and glyceryl monooleate (GMO). Using the binary matrix materials, NPs loaded with Sim (0–500?µg/mL) were prepared and showed an average particle size of about 150?nm. NP size stability was dependent on Sim concentration loaded in NPs. The suitability of NPs prepared with the binary matrix materials in Sim delivery for potential application in bone regeneration was supported by biocompatibility in pre-osteoclastic and pre-osteoblastic cells. Additional data demonstrated that biofunctional Sim was released from NPs that facilitated differentiation of osteoblasts (cells that form bones) while inhibiting differentiation of osteoclasts (cells that resorb bones). The overall work demonstrated the preparation of NPs from Ewax/GMO blends and characterization to ascertain potential suitability in Sim delivery for bone regeneration. Additional studies on osteoblast and osteoclast functions are warranted to fully evaluate the efficacy of Sim-loaded Ewax/GMO NPs using in-vitro and in-vivo approaches.     

Bacteriolysis by vancomycin-conjugated acryl nanoparticles and morphological component analysis

Nanoparticles are currently attracting considerable attention because of their ability to conjugate to various substances. As such, these nanoparticles can assist the transfer of the conjugated substance to target tissues where they are gradually released. In this study, vancomycin-conjugated nanoparticles (VCM NPs) were prepared. The antibacterial activity of VCM NPs was compared with that of VCM alone by exposure to vancomycin-resistant enterococci (VRE). The morphology of the cells was then analyzed by electron microscopy. VCM NPs were found to have more potent antibacterial activity against VRE compared to VCM alone, but the activity against vancomycin-sensitive enterococci (VSE) remained the same. The antibacterial activity of nanoparticles alone was the same against VSE and VRE. The nanoparticles were found to induce characteristic morphological changes in the bacteria based on scanning and transmission electron microscopy. The results suggest that the strong antibacterial activity of VCM NPs against VRE may be attributable not only to the well-known control release carrier property of the nanoparticles but to an additional mechanism that involves VCM NPs avoiding the drug resistant mechanism of VRE.