464256 Self-Assembled Di-Block Polymersomes As Artificial Immune Cells
Global healthcare in its current reactionary format is irrepressibly overwhelmed by a surplus of patients and tremendous medical expenses; associated treatment strategies are progressively insufficient. A paradigm shift centered on nanotechnology is anticipated to enable the gradual transition from reactionary to predictive medicine needed to enhance global wellness. In particular, it is hypothesized that the integration of synthetic immune cell mimics together with modern medical intervention will substantiate the concept of a smart vaccine capable of coordinating aggressive phagocyte and B-cell activities. Complex cellular systems, including relevant chemical accessories, are simulated through the design of smart polymeric materials called polymersomes (Ps). Ps are amphiphilic block copolymers that self-assemble into artificial vesicles via the hydrophobicity interactions of admixed aqueous and organic substances. The objective of the current study is to fabricate synthetic immune cells by exploiting tunable Ps membrane properties. The intelligent functionalization and digitization of Ps is postulated to mimic consummate immune cell behavior. Strategies are devised to supply an artificial vesicle affixed by several classes of antibodies and other species, including isolated mitochondria.
Successful functionalization requires the development of a bottom-up approach initiated through an assessment of the chemical bonds associating polymersome membrane properties with conjugated species and bacterial or viral shells. For the current application, chemical viability approximations suggest that a methoxypoly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA) diblock copolymer will be an ideal Ps prototype constituent1. The occurrence of polyethylene glycol within the hydrophilic block circumvents the probability of an Òautoimmune effectÓ in which artificial immune cells are dismantled by the bodyÕs natural defenses. The PDLLA block confers conditional stability to Ps stored outside the body at 4 ¡C and activity to those at physiological conditions.
A modified stirred-injection routine was applied for the self-assembly of Ps nanocarriers embedded with immunoglobulins, mitochondria, and the enzymatic species responsible for facilitating controlled Ps rupture. An organic solvent with a preferential capacity for generating nano-sized vesicles (i.e. tetrahydrofuran) was used to dissolve select block copolymers, before being infused with addendum species, including biotinylated homing devices. Hydration of the solution was achieved by the staggered addition of the organic solution to an aqueous mixture containing PBS, water, and other hydrophilic species, such as mitochondria. Theoretically, interfacial tensile forces between the hydrophobic regime and the hydration agent initiate spontaneous enclosure of the coupled Ps. Strategies were next devised to supply dynamic Ps that were conjugated with IgG primary antibodies along the peripheral hydrophilic regime and mitochondrial protein within the aqueous core. The direct introduction of IgG antibodies to a plain Ps solution yielded poor chemical attachment. Sufficient functionalization was achieved via a stable non-covalent conjugation associating the glycoprotein avidin with the water-soluble vitamin biotin. Tresylated polymer was subjected to an NMR chemical verification study prior to biotinylation in a biotin-lysine (biocytin) and methanol solution. The biotinylated Ps were washed in a secondary dialysis step that eliminated excess biocytin and organic solvent from the solution of interest.
The resulting vesicles were incubated overnight with an excess of avidin-rhodamine isothiocyanate before being dialyzed to remove free avidin from the Ps solution. Confocal microscopy images provide a validation of bilayer formation in addition to an optical quantification of the degree of avidin-biotin conjugation. Once avidin-biotin binding efficiencies were determined, unconjugated-avidin and biocytin functionalized Ps were incubated in a mixture of biotinylated IgG antibodies from human serum, before being dialyzed and introduced to a solution containing fluorescein isothiocyanate (FITC)-labeled mouse anti-human secondary antibodies. A final dialysis step ensured the exclusion of free immunoglobulins from solution. The relative degree of IgG surface attachment was measured by visual inspection of confocal microscopy images.
As part of a complementary Ps functionalization strategy, mitochondria were isolated from human dermal fibroblasts (HDF) via a rigorous centrifugation and homogenization routine. 120×106 HDFs were seeded 3 days prior to homogenization and fractionation. Structural integrity was confirmed using transmission electron microscopy (TEM) characterization. A subsequent Ps conjugation entailed the incorporation of mitochondria into the aqueous corona and catalase enzyme into the hydrophobic compartment prior to bilayer formation. Ps diameter, conventionally on the order of 100 nm, was tuned to accommodate for relatively large mitochondrial dimensions. Future bacterial and cytotoxicity assays will measure the viability of Ps that are functionalized with mitochondria and/or antibodies along the hydrophilic regime.
Physiochemical size distributions of plain Ps were obtained using transmission electron microscopy (TEM). A droplet saturated with Ps was uniformly coated on a 300-mesh copper-coated carbon grid, allowed to settle for 1 minute, and dried in ambient temperature conditions. Negative staining with a 1.5% uranyl acetate solution enabled differentiation of Ps edges and features. TEM images at 2000× magnification revealed spherical Ps of about 80 nm – 150 nm in diameter (Fig. 1). The successful generation of enclosed Ps was superseded by a rigorous mitochondrial isolation procedure.
A mitochondrial isolation routine was implemented on HDFs, and 500 μL of solution containing a pelleted sample were pre-fixed in 2.5% glutaraldehyde and maintained at 4 ¡C over a 48-hour time frame. The pellet was carefully removed from the fixing solution and sliced into six sections as a means of expanding surface area exposure and increasing the possibility of mitochondrial visibility. The samples were washed 2× in 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide. As part of an ethanol gradient dehydration, samples were suffused in 30% × 1, 50% × 1, 70% × 1, 85% × 1, 95% × 1, and 100% × 2 ethanol, respectively. The samples were immersed in a custom resin before being embedded via a 48-hour heat treatment and thin sectioned. A 300-mesh copper-coated carbon grid was coated with the sample, which was negatively stained using 1.5% uranyl acetate. TEM of the samples revealed sufficient mitochondrial yield for incorporation into the Ps aqueous corona. Future work will focus on the integration of these two structural elements. Fig. 2 shows a TEM image obtained at 15000× magnification of two neighboring mitochondria having diameters ranging from 400 nm – 450 nm. Fig. 3 shows a TEM obtained at 20000× magnification of a longitudinally oriented 700 nm long mitochondrion.
Experimental strategies were successfully implemented for the generation of Ps nanostructures and the isolation of mitochondrial protein. Extensive studies on the chemical nature of IgG surface attachment to an mPEG-PDLLA polymer will enable the design of Ps bounded externally by select antibodies. Specifically, surface functionalization is achieved exploiting high affinity avidin-biotin interactions. The terminal hydroxyl of the outer hydrophilic block is chemically modified through the application of a tresylation routine resolved by Nilsson and Mosbach2 in 1984. Tresylation is accompanied by a substitution reaction in which the organic sulfonyl chloride 2, 2, 2 – trifluoroethanesulfonyl chloride (tresyl chloride) converts the hydroxyl group of the terminal Ps hydrophilic block into a good sulfonate leaving group. Nucleophilic biotin (and its derivatives) can subsequently react with the resulting Ps, forming stable linkages. Integration of polymersomes with isolated mitochondria and IgG antibodies is anticipated to contribute to delayed bacterial growth and minimal cytotoxicity. Size-distribution and morphology of resulting particles were assessed using TEM and DLS measurements.
In an alternate design scheme, the internal projection of variable-size immunoglobulins is conceived to diminish the bulkiness of the final construct while conferring plasma cell functionality. Biotinylated antibodies may be conjugated to the internal peripheral hydrophilic regime with the utility of the previously prescribed avidin-biotin interaction. Precise chemical triggers within the vasculature will prompt the inversion and self-assembly of relevant response architectures. The disintegration mechanism is followed by extrusion of the diseased composite from the organism through natural secretions. One mechanism for the controlled antibody release postulates the incorporation of mitochondria into the aqueous Ps core and catalase enzyme into the hydrophobic corona. Recent studies conducted by Jang et al.3 have shown that catalase-embedded Ps may be engineered to release therapeutic agents in the presence of reactive oxygen species (ROS). The incorporation of isolated mitochondria into the aqueous Ps center will enable the localized production of hydrogen peroxide via the electron transport chain. Hydrogen peroxide will successively degrade into ROS and contribute to controlled Ps rupture.
Results of the prevalent analysis may be applied for the conception of a smart colloidal structure appended by several classes of homing devices. The emerging nano-structure is expected to drastically enhance the natural mechanisms associated with somatic hypermutation. Clinical injection of the composite Ps solution is hypothesized to activate an aggressive B-cell response in which thousands of antibodies disperse and mark antigens for destruction. Associated mitochondrial interactions are expected to drive controlled antibody release mechanisms. Proximate electrophysical studies will enable a self-powered device adept at sensing and tracking infected regions autonomously. Automated design is anticipated to permit the fabrication of a nano-device with response efficiencies and capabilities that exceed the healing properties shown by innate immune cells. The shift from reactionary to predictive intervention will prompt a new era in which early disease detection and treatment will contribute to enhanced longevity and productivity among patients.
1. Geilich, B. et al. Nanoscale, 2015; 7:3511.
2. Nilsson K, Mosbach K. Methods Enzymol. 1984;104:56-69.
3. Jang, W-S. Soft Matter, 2016,12, 1014-1020
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