We demonstrate that synthetic soft materials can extend the utility of natural vesicles, from predominantly hydrophilic reservoirs to functional colloidal carriers that facilitate the biomedical application of large aqueous-insoluble compounds. Near-infrared (NIR)-emissive polymersomes (50-nm-to 50-m-diameter polymer vesicles) were generated through cooperative self assembly of amphiphilic diblock copolymers and conjugated multi(porphyrin)-based NIR fluorophores (NIRFs). When compared with natural vesicles comprised of phospholipids, polymersomes were uniquely capable of incorporating and uniformly distributing numerous large hydrophobic NIRFs exclusively in their lamellar membranes. Within these sequestered compartments, long polymer chains regulate the mean fluorophore-fluorophore interspatial separation as well as the fluorophore-localized electronic environment. Porphyrin-based NIRFs manifest photophysical properties within the polymersomal matrix akin to those established for these high-emission dipole strength fluorophores in organic solvents, thereby yielding uniquely emissive vesicles. Furthermore, the total fluorescence emanating from the assemblies gives rise to a localized optical signal of sufficient intensity to penetrate through the dense tumor tissue of a live animal. Robust NIR-emissive polymersomes thus define a soft matter platform with exceptional potential to facilitate deep-tissue fluorescence-based imaging for in vivo diagnostic and drug-delivery applications.porphyrin ͉ vesicles ͉ nanoscale ͉ diblock copolymer S upramolecular self assembly has revolutionized soft materials research by enabling the efficient and high-throughput fabrication of complex multicomponent nanostructures (1-3). For decades, self-assembled vesicles comprised of phospholipids (liposomes) or small-molecule surfactants (4) have been used for sequestering high concentrations of hydrophilic compounds (5) and controlling their temporal release and distribution for maximal therapeutic efficacy (6). More recently, amphiphilic peptides and polymers have been shown to form very elaborate architectures (7-9) and serve as useful nanocontainers in aqueous solution (10). In particular, self-assembled materials are ideal for carrying promising imaging and therapeutic agents whose biomedical utility has hitherto been hampered by inadequate aqueous solubility (11). Here, we demonstrate the unique ability of synthetic amphiphiles to assemble into functional vesicles that membrane-disperse numerous large hydrophobic fluorophores and enable their specialized application for deeptissue fluorescence-based in vivo imaging.Although visible probes enable exquisite imaging of live animals by intravital microscopy (12), their utility is significantly limited at greater than submillimeter tissue depths as a result of extensive light scattering and optical absorption. Because light scattering diminishes with increasing wavelength, and hemoglobin electronic and water vibrational overtone absorptions approach their nadir over the near-infrared (N...

Liposomes (nontoxic/nonantigenic vesicles derived from phospholipids) have long been utilized in numerous biotechnology and pharmaceutical applications to improve therapeutic indices and enhance cellular uptake.1 Their structural stability, however, is dependent upon many intrinsic and environmental parameters that often serve to compromise their efficacy.2 Polymersomes (polymer vesicles formed from a wide variety of fully synthetic amphiphiles) 3 -5 have similar utility to their lipid counterparts but possess several advantageous properties including vastly superior stability6 and diverse functionality afforded by tuning material chemistries through polymer synthesis. Recently, there has been considerable interest in the development of degradable polymersomes suitable for in vivo drug delivery. Here, we present the generation of self-assembled vesicles comprised entirely of an amphiphilic diblock copolymer of poly(ethylene oxide) (PEO) and polycaprolactone (PCL), two previously FDAapproved polymers. Unlike degradable polymersomes formed from blending bioinert and hydrolyzable components,7 , 8 PEO-b-PCL-based vesicles promise to be fully bioresorbable, 9 leaving no potentially toxic byproducts upon their degradation. Moreover, unlike published reports of other degradable (peptide-, polyester-, or polyanhydride-based) polymersomes, 10-12 these bioresorbable vesicles are formed through spontaneous self-assembly of their pure component amphiphile.Poly(ethylene oxide) was chosen as the hydrophilic block as it imparts to the vesicle's surface biocompatibility and prolonged blood circulation times.13 -15 Polycaprolactone constitutes the vesicles' hydrophobic membrane portion. PCL is degraded by hydrolysis of its ester linkages in physiological conditions (such as in the human body) and has therefore received a great deal of attention for use as an implantable biomaterial in drug delivery devices, bioresorbable sutures, adhesion barriers, and scaffolds for injury repair via tissue engineering. 16 -19 Compared to other biodegradable aliphatic polyesters, PCL has several advantageous properties, including (1) high permeability to small drug molecules, (2) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript neutral pH environment upon degradation, (3) facility in forming blends with other polymers, and (4) suitability for long-term delivery afforded by slow erosion kinetics as compared to polylactide (PLA), polyglycolide (PGA), and polylactic-co-glycolic acid (PLGA). 17 Utilization of PCL as the hydrophobic block in our formulations promises that the resultant polymersomes should have safe and complete in vivo degradation.Amphiphilic poly(ethylene oxide)-b-polycaprolactone was generated via ring-opening polymerization of cyclic ∊-caprolactone (CL) in the presence of stannous(II) octoate (SnOct) and monocyano-or monomethoxypoly(ethylene oxide) (PEO, 0.75K, 1.1K, 1.5K, 2K, 5K, 5.5K, 5.8K; Polymer Source, Dorval, Canada). 20 The reactions yielded PEO-b-PCL copolymers with varying PCL block size (betwe...

Dendritic cells (DCs) play a pivotal role in both immune tolerance and the initiation of immunological responses. The ability to track DCs in vivo is imperative for the development of DC-based cellular therapies and to advance our understanding of DC function and pathophysiology. Here, we conjugate a cell permeable peptide, Tat, to near-infrared (NIR) emissive polymersomes in order to enable efficient intracellular delivery for future DC tracking with these optical probes. NIR imaging allows quantitative, repetitive, in vivo detection of fluorophore-laden cells, at centimeter tissue depths without disturbing cellular function. Flow cytometry and confocal microscopy results indicate that Tat-mediated polymersome delivery to DCs is concentration and time dependent, resulting in punctate intracellular localization. Further, loading cells with Tat NIR emissive polymersomes does not interfere with cytokine-induced DC maturation and has modest effects on DC viability, but has a significant effect on mature DC-induced activation of naive T cells. We observe significant uptake of NIR emissive polymersomes when conjugated to the peptide, with a lower detection limit of 5000 labeled DCs. The extent of polymersome delivery is estimated as 70 000 +/- 10 000 vesicles/cell, equivalent to 0.7 +/- 0.1 fmol of NIR fluorophore. Our studies will enable future in vivo tracking of ex vivo labeled DCs by NIR fluorescence based imaging.