Self-assembled stimuli-responsive polyrotaxane core-shell particles.

Thermodynamically assembled core-shell nanocarriers are potential candidates for drug delivery applications due to their submicrometer size and the ability to load drugs into their hydrophobic core. Herein, we describe the formation of core-shell particles that consist of noncovalent polymers, that is, polyrotaxanes (PRXs), that form an α-cyclodextrin (αCD) core surrounded by a corona of low-fouling poly(ethylene glycol) (PEG). The PRX core-shell particles are able to sequester small organic molecules, such as pyrene and calcein, releasing these small molecules during degradation. The small, cellular peptide, glutathione, was used to degrade the particles through the reductive cleavage of disulfide bonds that stabilize the individual PRX polymers. Cleavage of a single bond allows for the degradation of the supramolecular-polymer, making these PRX core-shell particles highly responsive. Furthermore, these particles demonstrate negligible cytotoxicity in mammalian cells, making them promising carriers for future drug delivery research.

Bio-click chemistry: enzymatic functionalization of PEGylated capsules for targeting applications.

All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.

Targeting of cancer cells using click-functionalized polymer capsules.

Targeted delivery of drugs to specific cells allows a high therapeutic dose to be delivered to the target site with minimal harmful side effects. Combining targeting molecules with nanoengineered drug carriers, such as polymer capsules, micelles and polymersomes, has significant potential to improve the therapeutic delivery and index of a range of drugs. We present a general approach for functionalization of low-fouling, nanoengineered polymer capsules with antibodies using click chemistry. We demonstrate that antibody (Ab)-functionalized capsules specifically bind to colorectal cancer cells even when the target cells constitute less than 0.1% of the total cell population. This precise targeting offers promise for drug delivery applications.

Monoclonal antibody-functionalized multilayered particles: targeting cancer cells in the presence of protein coronas.

Engineered particles adsorb biomolecules (e.g., proteins) when introduced in a biological medium to form a layer called a "corona". Coronas, in particular the protein corona, play an important role in determining the surface properties of particles and their targeting abilities. This study examines the influence of protein coronas on the targeting ability of layer-by-layer (LbL)-assembled polymer capsules and core-shell particles functionalized with monoclonal antibodies. Upon exposure of humanized A33 monoclonal antibody (huA33 mAb)-functionalized poly(methacrylic acid) (PMA) capsules or huA33 mAb-PMA particles to human serum, a total of 83 or 65 proteins were identified in the protein coronas, respectively. Human serum of varying concentrations altered the composition of the protein corona. The antibody-driven specific cell membrane binding was qualitatively and quantitatively assessed by flow cytometry and fluorescence microscopy in both the absence and presence of a protein corona. The findings show that although different protein coronas formed in human serum (at different concentrations), the targeting ability of both the huA33 mAb-functionalized PMA capsules and particles toward human colon cancer cells was retained, demonstrating no significant difference compared with capsules and particles in the absence of protein coronas: ∼70% and ∼90% A33-expressing cells were targeted by the huA33 mAb-PMA capsules and particles, respectively, in a mixed cell population. This result demonstrates that the formation of protein coronas did not significantly influence the targeting ability of antibody-functionalized LbL-polymer carriers, indicating that the surface functionality of engineered particles in the presence of protein coronas can be preserved.

Particles on the move: intracellular trafficking and asymmetric mitotic partitioning of nanoporous polymer particles.

Nanoporous polymer particles (NPPs) prepared by mesoporous silica templating show promise as a new class of versatile drug/gene delivery vehicles owning to their high payload capacity, functionality, and responsiveness. Understanding the cellular dynamics of such particles, including uptake, intracellular trafficking, and distribution, is an important requirement for their development as therapeutic carriers. Herein, we examine the spatiotemporal map of the cellular processing of submicrometer-sized disulfide-bonded poly(methacrylic acid) (PMASH) NPPs in HeLa cells using both flow cytometry and fluorescence microscopy. The data show that the PMASH NPPs are transported from the early endosomes to the lysosomes within a few minutes. Upon cell division, the lysosome-enclosed PMASH NPPs are distributed asymmetrically between two daughter cells. Statistical analysis of cells during cytokinesis suggests that partitioning of particles is biased with an average segregation deviation of 60%. Further, two-dimensional difference gel electrophoresis (2D-DIGE) analysis reveals that 127 out of 3059 identified spots are differentially regulated upon exposure to the PMASH NPPs. Pathway analysis of the proteomics data suggests that ubiquitylation, a reversible modification of cellular proteins with ubiquitin, plays a central role in overall cellular responses to the particles. These results provide important insights into the cellular dynamics and heterogeneity of NPPs, as well as the mechanisms that regulate the motility of these particles within cells, all of which have important implications for drug susceptibility characteristics in cancer cells using particle-based carriers.

Differential roles of the protein corona in the cellular uptake of nanoporous polymer particles by monocyte and macrophage cell lines.

Many biomolecules, mainly proteins, adsorb onto polymer particles to form a dynamic protein corona in biological environments. The protein corona can significantly influence particle-cell interactions, including internalization and pathway activation. In this work, we demonstrate the differential roles of a given protein corona formed in cell culture media in particle uptake by monocytes and macrophages. By exposing disulfide-stabilized poly(methacrylic acid) nanoporous polymer particles (PMASH NPPs) to complete cell growth media containing 10% fetal bovine serum, a protein corona, with the most abundant component being bovine serum albumin, was characterized. Upon adsorption onto the PMASH NPPs, native bovine serum albumin (BSA) was found to undergo conformational changes. The denatured BSA led to a significant decrease in internalization efficiency in human monocytic cells, THP-1, compared with the bare particles, due to reduced cell membrane adhesion. In contrast, the unfolded BSA on the NPPs triggered class A scavenger receptor-mediated phagocytosis in differentiated macrophage-like cells (dTHP-1) without a significant impact on the overall internalization efficiency. Taken together, this work demonstrates the disparate effects of a given protein corona on particle-cell interactions, highlighting the correlation between protein corona conformation in situ and relevant biological characteristics for biological functionalities.

Dynamic culturing of smooth muscle cells in tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering.

Porous, tubular, flexible, and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm and inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 µm and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a microporous outer layer of 0.2 mm with an average pore size of 28 µm and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, and average wall shear rate 320 s(-1)). Although SMCs proliferated under both conditions, cell numbers were three to five times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also, in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular poly(trimethylene carbonate) scaffolds.

Uptake and intracellular fate of disulfide-bonded polymer hydrogel capsules for Doxorubicin delivery to colorectal cancer cells.

Understanding the interactions between drug carriers and cells is of importance to enhance the delivery of therapeutics. The release of therapeutics into different intracellular environments, such as the lysosomes or the cell cytoplasm, will impact their pharmacological activity. Herein, we investigate the intracellular fate of layer-by-layer (LbL)-assembled, submicrometer-sized polymer hydrogel capsules in a human colon cancer derived cell line, LIM1899. The cellular uptake of the disulfide-stabilized poly(methacrylic acid) (PMA(SH)) capsules by colon cancer cells is a time-dependent process. Confocal laser scanning microscopy and transmission electron microscopy reveal that the internalized capsules are deformed in membrane-enclosed compartments, which further mature to late endosomes or lysosomes. We further demonstrate the utility of these redox-responsive PMA(SH) capsules for the delivery of doxorubicin (DOX) to colon cancer cells. The DOX-loaded PMA(SH) capsules demonstrate a 5000-fold enhanced cytotoxicity in cell viability studies compared to free DOX.