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.

Size and rigidity of cylindrical polymer brushes dictate long circulating properties in vivo.

Studies of spherical nanoengineered drug delivery systems have suggested that particle size and mechanical properties are key determinants of in vivo behavior; however, for more complex structures, detailed analysis of correlations between in vitro characterization and in vivo disposition is lacking. Anisotropic materials in particular bear unknowns in terms of size tolerances for in vivo clearance and the impact of shape and rigidity. Herein, we employed cylindrical polymer brushes (CPBs) to answer questions related to the impact of size, length and rigidity on the in vivo behavior of PEGylated anisotropic structures, in particular their pharmacokinetics and biodistribution. The modular grafting assembly of CPBs allowed for the systematic tailoring of parameters such as aspect ratio or rigidity while keeping the overall chemical composition the same. CPBs with altered length were produced from polyinitiator backbones with different degrees of polymerization. The side chain grafts consisted of a random copolymer of poly[(ethylene glycol) methyl ether methacrylate] (PEGMA) and poly(glycidyl methacrylate) (PGMA), and rendered the CPBs water-soluble. The epoxy groups of PGMA were subsequently reacted with propargylamine to introduce alkyne groups, which in turn were used to attach radiolabels via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). Radiolabeling allowed the pharmacokinetics of intravenously injected CPBs to be followed as well as their deposition into major organs post dosing to rats. To alter the rigidity of the CPBs, core-shell-structured CPBs with polycaprolactone (PCL) as a water-insoluble and crystalline core and PEGMA-co-PGMA as the hydrophilic shell were synthesized. This modular buildup of CPBs allowed their shape and rigidity to be altered, which in turn could be used to influence the in vivo circulation behavior of these anisotropic polymer particles. Increasing the aspect ratio or altering the rigidity of the CPBs led to reduced exposure, higher clearance rates, and increased mononuclear phagocytic system (MPS) organ deposition.

Engineering cellular degradation of multilayered capsules through controlled cross-linking.

We report a versatile approach for controlling the intracellular degradation of polymer capsules by tailoring the degree of cross-linking in the capsules. Poly(2-diisopropylaminoethyl methacrylate) capsules were assembled by the layer-by-layer technique and covalently stabilized with a redox-responsive bisazide cross-linker using click chemistry. The degree of cross-linking, determined using radiation scintillation counting, was tuned from 65% to 98% by adjusting the amount of cross-linker used to stabilize the polymer films. Transmission electron microscopy and fluorescence microscopy studies showed that the pH responsiveness of the capsules was maintained, regardless of the degree of cross-linking. Atomic force microscopy measurements on planar surfaces revealed that increasing the degree of cross-linking decreased the film roughness (from 8.7 to 1.7 nm), hence forming smoother films; however the film thicknesses were not significantly altered. Cellular studies showed that the rate of intracellular degradation of the capsules could be controlled between 0 and 6 h by altering the degree of cross-linking in the polymer capsules. These studies also demonstrated that the cellular degradation of highly cross-linked capsules (>90%) was significantly retarded compared to degradation in simulated cellular conditions. This suggests that the naturally occurring cellular reducing environment is rapidly depleted, and there is a significant delay before the cells can replenish the reducing environment. The modular and versatile nature of this approach lends itself to application to a wide range of polymer carriers and thus offers significant potential for the design of polymer-based systems for drug and gene delivery.

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.

Low-fouling poly(N-vinyl pyrrolidone) capsules with engineered degradable properties.

We report the assembly of low-fouling polymer capsules with engineered deconstruction properties by using a combination of layer-by-layer (LbL) assembly and click chemistry. Preformed, hydrogen-bonded multilayers of alkyne-functionalized poly(N-vinyl pyrrolidone) (PVPON(Alk)) and poly(methacrylic acid) (PMA) assembled at pH 4 on silica particles were cross-linked with a bisazide linker (containing a disulfide link) through alkyne-azide click chemistry. Following dissolution of the silica template particles, and altering the solution pH to 7.2 to disrupt hydrogen bonding between PVPON(Alk) and PMA to effect removal of PMA, stable, cross-linked PVPON capsules were obtained. The presence of the disulfide bond in the bisazide linker endowed the PVPON capsules with degradable characteristics under model intracellular conditions. The capsules deconstructed within 4 h in the presence of 5 mM glutathione. The cross-linked PVPON(Alk) multilayers (assembled on silica particles) were low-fouling to a range of proteins, including fibrinogen, lysozyme, immunoglobulin G, and bovine serum albumin. Further, MTT assays showed that the PVPON capsules had no effect on the proliferation of cells from a human colon cancer cell line (LIM1899), indicating negligible cytotoxicity toward the LIM1899 cells. The low-fouling, degradable, and low cytotoxicity characteristics of the PVPON capsules makes them attractive as a platform for the development of advanced therapeutic delivery systems.