(256v) Self-Assembled Di-Block Polymersomes As Artificial Immune Cells

Introduction

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.

Methods

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.

Results

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.

Discussion

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.

Literature Citations

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