This paper reports measurements of particle-wall and particle-particle interactions in levitated colloidal ensembles using integrated total internal reflection microscopy (TIRM) and video microscopy (VM) techniques. In levitated colloidal ensembles with area fractions of phiA = 0.03-0.25, ensemble TIRM measured height distribution functions are used to interpret particle-wall interactions, and VM measured pair distribution functions are used to interpret particle-particle interactions using inverse Ornstein-Zernike (OZ) and three-dimensional inverse Monte Carlo (MC) analyses. An inconsistent finding is the observation of an anomalous long-range particle-particle attraction and recovery of the expected Derjaguin-Landau-Verwey-Overbeek (DLVO) particle-wall interactions for all concentrations examined. Because particle-wall and particle-particle potentials are expected to be consistent in several respects, the analytical and experimental methods employed in this investigation are examined for possible sources of error. Comparison of inverse OZ and three-dimensional inverse MC analyses are used to address uncertainties related to dimensionality, effects of particle concentration, and assumptions of the OZ theory and closure relations. The possible influence of charge heterogeneity and particle size polydispersity on measured distribution functions is discussed with regard to inconsistent particle-wall and particle-particle potentials. Ultimately, achieving a consistent understanding of particle-wall and particle-particle interactions in interfacial and confined colloidal systems is essential to numerous complex fluid and advanced material technologies.
Polymersomes were functionalized using azide-alkyne ''click'' chemistry with two targeting peptides: GRGDSP and the recently designed fibronectin mimetic peptide, PR_b, which has been shown to bind with high specificity to the a 5 b 1 integrin expressed on colon cancer cells. The ability of these peptide functionalized polymersomes to achieve targeted delivery to colon cancer cells was assessed by studying their delivery to CT26.WT and Caco-2 cells in vitro. The diblock copolymer poly(ethylene oxide)-b-poly (1,2-butadiene) was synthesized and self-assembled to form polymersomes, which were subsequently functionalized with peptides using a ''click'' conjugation reaction. Drug delivery efficacy of these peptide functionalized polymersomes loaded with fluorescent markers or the chemotherapeutic, doxorubicin, was assessed and compared. In addition, the cell binding and internalization of fluorescently labeled polymersomes was imaged using confocal microscopy. PR_b functionalized polymersomes are found to significantly outperform both GRGDSP and non-functionalized polymersomes, both in terms of promoting cell binding and internalization and doxorubicin cytotoxicity. Moreover, PR_b functionalized polymersomes are found to act as highly target specific drug delivery agents, thus highlighting them as a promising model targeted drug delivery system.
Single-tailed peptide-amphiphiles have been shown to form nanofibers in solution and gel after screening of their electrostatic charges, and those containing cell-binding motifs are promising as tissue engineering scaffolds. A fibronectin-mimetic peptide sequence was developed, containing both the primary binding domain RGD and the synergy binding domain PHSRN, which has shown superior cell adhesion properties over simple RGD sequences and fibronectin in 2D culture. In order to test this sequence in a 3D environment in the future, we have designed a C(16) single-tailed peptide-amphiphile, PR_g (with a peptide headgroup of GGGSSPHSRN(SG)(5)RGDSP), that forms nanofibers and a gel in solution without any screening of its positive charge. In this study, we characterized the self-assembly properties of the PR_g peptide-amphiphile via critical micelle concentration (CMC) measurements, circular dichroism (CD) spectroscopy, cryo-transmission electron microscopy (cryo-TEM), small angle neutron scattering (SANS), and rheology measurements. The CMC of the PR_g amphiphile was determined to be 38 microM. CD measurements showed that even though the peptide formed an unordered secondary structure, the peptide-amphiphile's spectrum after aging resembled more the spectrum of an alpha+beta protein. Cryo-TEM images of a 100 microM peptide-amphiphile solution showed individual nanofibers with a diameter of approximately 10 nm and lengths on the order of several micrometers. Images taken at higher concentrations (1 mM) show a high degree of bundling among the nanofibers, and at even higher concentrations (3 and 4 mM) SANS measurements also indicated that the peptide-amphiphile formed rod-shaped structures in solution. The peptide-amphiphile gel was monitored by parallel-plate rheometry, and the elastic modulus (G') was greater than the viscous modulus (G''), which indicates that PR_g forms a gel. The shear modulus for a 2 day old gel was measured to be approximately 500 Pa, which is within the modulus range for living tissue; thus, the PR_g gel shows potential as a possible scaffold for tissue engineering.
Polymersomes, polymeric vesicles that self-assemble in aqueous solutions from block copolymers, have been avidly investigated in recent years as potential drug delivery agents. Past work has highlighted peptide-functionalized polymersomes as a highly promising targeted delivery system. However, few reports have investigated the ability of polymersomes to operate as gene delivery agents. In this study, we report on the encapsulation and delivery of siRNA inside of peptide-functionalized polymersomes composed of poly(1,2-butadiene)-b-poly(ethylene oxide). In particular, PR_b peptide-functionalized polymer vesicles are shown to be a promising system for siRNA delivery. PR_b is a fibronectin mimetic peptide targeting specifically the α(5)β(1) integrin. The Orai3 gene was targeted for siRNA knockdown, and PR_b-functionalized polymer vesicles encapsulating siRNA were found to specifically decrease cell viability of T47D breast cancer cells to a certain extent, while preserving viability of noncancerous MCF10A breast cells. siRNA delivery by PR_b-functionalized polymer vesicles was compared to that of a current commercial siRNA transfection agent, and produced less dramatic decreases in cancer cell viability, but compared favorably in regards to the relative toxicity of the delivery systems. Finally, delivery and vesicle release of a fluorescent encapsulate by PR_b-functionalized polymer vesicles was visualized by confocal microscopy, and colocalization with cellular endosomes and lysosomes was assessed by organelle staining. Polymersomes were observed to primarily release their encapsulate in the early endosomal intracellular compartments, and data may suggest some escape to the cytosol. These results represent a promising first generation model system for targeted delivery of siRNA.
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