Benchmarking bicontinuous nanospheres against polymersomes for

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Biological and Medical Applications of Materials and Interfaces

Benchmarking bicontinuous nanospheres against polymersomes for in vivo biodistribution and dual intracellular delivery of lipophilic and water soluble payloads Sean David Allen, Sharan Bobbala, Nicholas Karabin, Mallika Modak, and Evan Alexander Scott ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09906 • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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ACS Applied Materials & Interfaces

Benchmarking bicontinuous nanospheres against polymersomes for in vivo biodistribution and dual intracellular delivery of lipophilic and water soluble payloads Sean D. Allen1†, Sharan Bobbala2†, Nicholas B. Karabin2, Mallika Modak2, and Evan A. Scott1,2,3,4,5* 1

Interdisciplinary Biological Sciences, Northwestern University, Evanston, USA.

2

Department of Biomedical Engineering, Northwestern University, Evanston, USA.

3

Chemistry of Life Processes Institute, Northwestern University, Evanston, USA.

4

Simpson Querrey Institute, Northwestern University, Chicago, USA.

5

Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, USA.

†

These authors contributed equally to this work.

* Corresponding Author: [email protected]

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Abstract Bicontinuous nanospheres (BCNs) are polymeric analogues to lipid cubosomes, possessing cubic liquid crystalline phases with high internal surface area, aqueous channels for loading hydrophilic molecules, and high hydrophobic volume for lipophilic payloads. Primarily due to difficulties in scalable and consistent fabrication, neither controlled delivery of payloads via BCNs nor their organ or cellular biodistributions following in vivo administration have been demonstrated or characterized. We have recently validated flash nanoprecipitation as a rapid method of assembling uniform monodisperse 200-300 nm diameter BCNs from poly(ethylene glycol)-b--b-poly(propylene sulfide) (PEG-b-PPS) copolymers. Here, we compare these BCNs both in vitro and in vivo to 100 nm PEG-b-PPS polymersomes (PSs), which have been wellcharacterized as nanocarriers for controlled delivery applications. Using a small molecule fluorophore and a fluorescently tagged protein as respective lipophilic and water soluble model cargos, we demonstrate that BCNs can achieve significantly higher encapsulation efficiencies for both payloads on a per unit mass basis. At timepoints of 4 h and 24 h after intravenous administration to mice, we found significant differences in organ-level uptake between BCNs and PSs, with BCNs showing reduced accumulation in the liver and increased uptake in the spleen. Despite these organ-level differences, BCNs and PSs displayed strikingly similar uptake profiles by immune cell populations in vitro and in the liver, spleen, and blood, as assayed by flow cytometry. In conclusion, we have found PEG-b-PPS BCNs to be well suited for dual loading and delivery of molecular payloads, with a favorable organ biodistribution and high cell uptake by therapeutically relevant immune cell populations. Keywords:

nanoparticle,

self-assembly,

flash

nanoprecipitation,

bicontinuous nanospheres, polymersome


biodistribution,

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1. Introduction Polymeric nanocarriers are highly customizable platforms for the stable transport of diverse molecular payloads to specific cell populations for controlled and bioresponsive intracellular delivery. Owing to their vesicular morphology, polymersomes (PSs) are a polymeric nanocarrier that present unique advantages for the simultaneous loading and delivery of both hydrophobic and hydrophilic compounds respectively within their lipophilic membranes and large aqueous lumens(1). Since they are typically composed of long highly customizable polymers, polymersomes present numerous benefits over liposomes, including higher stability, increased ‘stealth’ characteristics, and greater amenability to chemical modification for triggered payload release and surface engineering for cell targeting(2). Polymeric bicontinuous nanospheres (BCNs) present similar advantages over their lipid analogs, cubosomes(3). These liquid crystalline nanostructures have a cubic internal organization of aqueous channels capable of loading hydrophilic compounds, much the same as PSs. While PSs show significant burst release characteristics for water soluble payloads, BCNs have demonstrated a more sustained and size-dependent release profile for loaded molecules due to the trapping of payloads within their interconnected narrow channels. BCNs also possess increased hydrophobic volume for loading lipophilic compounds compared to PSs(4). Despite their aforementioned advantages, BCNs remain undercharacterized and underutilized for controlled delivery, primarily due to difficulties in scalable and consistent fabrication. BCNs are often composed of complex copolymers that require multistep or complex syntheses(5-6), though pioneering work has been performing using simpler diblock copolymers(7). Furthermore, self-assembly of these polymers can result in mixtures of different and polydisperse nanostructures, with only a small proportion being BCNs. As a result, most research has focused on the novelty of the polymers used, rather than the potential biomedical applications of BCNs(5, 8-9). Notable recent exceptions have been the study of pyrene(10) and ibuprofen(11) release rates from BCNs with temperature-sensitive pores and an in vitro comparison of PSs and BCNs in their ability to stimulate dendritic cells to present antigen(12). None of these studies included in vivo experiments, and thus the demonstration and characterization of controlled delivery applications as well as organ or cellular biodistributions of BCNs is currently lacking. We have recently added to the short list of polymers capable of forming BCNs by demonstrating their rapid fabrication using poly(ethylene glycol)17-block-poly(propylene sulfide)75, (PEG17-b-PPS75)(12-13). This diblock copolymer, relatively simple in comparison to some of the



other BCN-forming polymers, was found to form BCNs when subjected to a formulation process known as flash nanoprecipitation (FNP). FNP is rapid, scalable, sterile, and capable of forming uniform PEG-b-PPS nanocarriers of numerous morphologies, including micelles, filomicelles, PSs, and BCNs, simply by varying the hydrophilic mass fraction of the polymer, with BCNs forming at low hydrophilic mass fractions (fPEG < 0.12 for PEG-b-PPS)(13). PEG-b-PPS is an oxidation-sensitive polymer; the sulfur in the propylene sulfide block reacts with reactive oxygen species, converting to more hydrophilic sulfoxide and sulfone derivatives and destabilizing the aggregate structure of the nanocarrier(14-15). This results in the bioresponsive or triggered delivery of loaded cargo under oxidative conditions, such as those within endolysosomal compartments of cells(16) or following irradiation in the presence of a photo-oxidizer(14-16). PEG-bPPS nanocarriers have demonstrated utility for in vivo applications involving the targeted delivery of payloads to immune cell populations, particularly antigen presenting cells that are critical targets during immunotherapy and vaccination(17-20). As PEG-b-PPS BCNs demonstrated the same negligible effects on cell viability as other PEG-b-PPS nanocarriers in vitro(12), we sought to further investigate whether the BCN morphology can enhance the loading and intracellular delivery of hydrophobic payloads as well as characterize their organ and cellularlevel biodistributions. For relevancy, our BCN measurements were benchmarked against PEGb-PPS PSs possessing the same surface chemistry, which we(13, 20) and others(14, 16, 18) have characterized extensively in vitro and in vivo.

2. Materials and Methods 2.1 Animal care and use C57BL/6J female mice, 8-10 weeks old, were purchased from Jackson Laboratories. All mice were house and maintained in the Center for Comparative Medicine at Northwestern University. All animal experimental procedures were performed according to protocols approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC). 2.2 Materials Unless explicitly stated below, all reagents and chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).


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2.3 Polymer Synthesis Poly(ethylene glycol)17-block-poly(propylene sulfide)36 and Poly(ethylene glycol)17-blockpoly(propylene sulfide)75 were synthesized as described previously(12-13). In brief, methyl ether PEG (MW: 750 Da) was end-functionalized with a mesylate and subsequently reacted with thioacetic acid. Base deprotection of the resulting thioacetate group afforded a thiolate anion, which was used for living ring opening polymerization of propylene sulfide to produce the diblock copolymer. 2.4

Formulation

of

polymersomes

and

bicontinuous

nanospheres

by

flash

nanoprecipitation FNP of PSs and BCNs was performed as previously described(12-13). Briefly, PEG-b-PPS copolymer (10 mg) was dissolved in 500 µL of THF along with the hydrophobic cargo and was loaded into a 1 mL syringe. Another 1 mL syringe was loaded with 500 µL of 1xPBS (for PSs) or water (for BCNs). These two solutions were impinged against one another in a hand-driven confined impingement jets (CIJ) mixer. For PSs, the impinged solution was re-divided between the two syringes for an additional 4 more impingements before finally being introduced to a reservoir of 1.5 mL of 1xPBS to decrease polydispersity as described previously(13). For BCNs, the impinged solution was immediately diluted with 1.5 mL of water. Unloaded formulations were dialyzed overnight in a 100 kDa Float-a-lyzer G2 device (Repligen, Waltham, MA, USA) in 1xPBS to remove residual organic solvent. For dual loaded PS and BCN formulations, hydrophobic DiD (5 µL from 2.5 mg/ml in THF, ThermoFisher) and hydrophilic FITC-BSA (100 µL from 2 mg/ml in water or PBS) were added to the THF and aqueous phase, respectively prior to the impingement. To ensure removal of both unloaded hydrophobic and hydrophilic dyes, formulations were column filtered using a Sepharose CL-6B column with PBS as an eluent. For in vivo experiments, PSs or BCNs were covalently labelled with maleimide-functionalized fluorescent dye (DyLight 755-maleimide). Solutions of PSs or BCNs (5 mg/mL) were added with 0.07 mM of DyLight 755-maleimide (Fisher), vortexed overnight at room temperature and excess unreacted dye was then removed by dialyzing against sterile PBS. Ethyl eosin loaded formulations were created using previously published modifications of the protocol described above(14-15). 2.5 Characterization of PS and BCN nanostructures



The diameters and polydispersity of PS and BCN formulations were determined via dynamic light scattering (DLS) using a Nano 300 ZS zetasizer (Malvern Panalytical, Malvern, UK). Particle concentrations were determined using nanoparticle tracking analysis (NTA) on a Nanosight NS300 (Malvern Panalytical, Malvern, UK). The morphology of PSs and BCNs were characterized using cryogenic transmission electron microscopy (cryoTEM). Briefly, 4 µL of sample (5 mg/mL) was applied to a pretreated holey carbon 400 mesh TEM grid and was plunge-frozen with a Gatan Cryoplunge freezer. These specimens were imaged using a JEOL 3200FS transmission electron microscope operating at 300 keV at 4000× nominal magnification. All the images were collected in vitreous ice using a total dose of ∼10 e− Å−2 and a nominal defocus range of 2.0–5.0 µm. BCNs were additionally characterized using negative staining TEM. PS and BCN samples were also characterized for overall aggregate morphology and internal structure using small angle x-ray scattering (SAXS). Small angle X-ray scattering (SAXS) studies were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) beamline at Argonne National Laboratory's Advanced Photon Source (Argonne, IL, USA) with 10 keV (wavelength λ = 1.24 Å) collimated X-rays, as described previously(12, 15, 21). Encapsulation efficiency was defined as the amount of cargo remaining in nanocarriers after removal of unloaded cargo, as a percentage of the total amount of cargo initially added to the formulation. Loading capacity was defined as the amount of loaded cargo after removal of unloaded cargo, as a weight percentage of the mass of the polymer used to form the nanocarriers. Ethyl eosin encapsulation efficiency was determined by comparing the fluorescence intensity of ethyl eosin loaded nanocarrier formulations (ex/em 525/560 nm) before and after filtration on a sepharose 6B column to remove unencapsulated ethyl eosin. The encapsulation efficiency of DiD and FITC-BSA was determined by fluorescence measurements using the excitation/emission of 644/670 and 495/519, respectively. Measurements were taken using a Spectromax M3 plate reader, using equal concentrations and volumes of PS and BCN formulations that were loaded with equal initial concentrations of ethyl eosin. DyLight 755 conjugation was assessed by measuring the fluorescence of 10 uL of PS or BCN formulation in 100 µL of 1xPBS (ex/em 755/770) after dialysis to remove conjugated dye. 2.6 Post Formulation Uptake of Pyrene 50 µL of BCN and PS formulations at copolymer concentrations of 1 mg/mL were added to 50 µL of pyrene (4 µM in a buffer of 1% tetrahydrofuran + 1% ethanol in 1xPBS) in glass-


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bottom black walled 96 well plates for time course measurements of pyrene fluorescence(22). Readings (excitation/emission of 331/390 nm) were taken on a Spectromax M3 immediately after the addition of nanocarrier formulations to pyrene solutions, and every subsequent 30 seconds for 1 h. 2.7 In Vitro Uptake of BCNs and PSs RAW 264.7 cells were seeded in non-tissue culture treated plastic 48 well plates at 100,000 cells per well. To these cells were added BCNs and PSs at equal polymer concentrations (0.5 mg/mL). Cells were incubated with FITC-BSA and DiD dual loaded BCNs or PSs for 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h before cells were collected for uptake analysis using flow cytometry. Cells were stained with Zombie Aqua cell viability dye (BioLegend) for 15 minutes at a 1:100 dilution in cell staining buffer (eBioscience), washed with 1xPBS, and briefly fixed with 2% paraformaldehyde prior to analysis on a BD LSR Fortessa. Flow cytometry data was analyzed using the online Cytobank analysis suite(23). 2.8 In Vivo Administration of BCNs and PSs 100 µL of 5 mg/mL formulations of BCNs and PSs were injected via the tail vein into C57BL/6J mice. 4 h or 24 h after injection, 500 µL of blood was collected retro-orbitally, and mice were euthanized. Liver, kidneys, lungs, and spleens were collected and imaged with an IVIS Spectrum in vivo imaging system (Perkin Elmer, Waltham, MA, USA). Organs were imaged using the preset filter combination for DyLight 755 and automated acquisition settings at the ‘B’ field of view and 0.5 mm height. Total radiant efficiency was calculated using Living Image software. Liver, blood, and spleens were retained for processing into single cell suspensions for flow cytometry. 2.9 Flow Cytometry for In Vivo PS and BCN Uptake by Immune Cell Populations Liver, blood, and spleen from mice 4 hand 24 h post intravenous (IV) administration of BCNs and PSs were processed for flow cytometry. Livers were mechanically dissected into 1 mm3 pieces, which were incubated in a digestion solution of 0.1 mg/mL DNase I (Roche) and 1.6 mg/mL Collagenase D (Roche) in serum-free RPMI (ThermoFisher) at 37 °C for 45 minutes while shaking at 500 rpm, with breaks every 15 minutes for rapid vortexing of the samples. The



liver cells were then passed through a 70 µm nylon mesh. Cells were spun at 100 rcf for 2 minutes twice, retaining the supernatant each time, which consisted of a single cell suspension of liver immune cells. Blood was spun at 3000 rpm for 25 minutes, and serum was removed from the cell pellet. Blood cells were lysed using ACK lysis buffer (Gibco) 3 times. Remaining cells were used for staining. Spleens were mechanically disrupted and passed through a 70 µm nylon mesh to make a single cell suspension. Single cell suspensions were stained for viability using Zombie Aqua (Biolegend), and were blocked using an anti-CD16/32 blocking antibody. Cells were then stained with a panel of antibodies for immune cell populations, all antibodies were from Biolegend unless specified: BUV396 anti-CD45 (BD), BV650 anti-IA-IE, BV711 anti-Ly-6C, BV605 anti-F4/80, FITC antiNK1.1, FITC anti-CD3, FITC anti-CD19, PerCP/Cy5.5 anti-CD11b, PE anti-B220, BV421 antiCD11c, APC anti-CD8a, and AlexaFluor 700 anti-Ly-6G. Flow cytometry was performed on a LSR Fortessa 6-laser instrument (BD), and data was analyzed using the online Cytobank analysis suite(23), using a gating strategy demonstrated in Supplemental Figure 5.

3. Results and Discussion 3.1 BCNs formed by FNP are monodisperse nanocarriers capable of high hydrophobic cargo loading We have previously reported the use of FNP to assemble diverse PEG-b-PPS nanostructure morphologies including micelles, filomicelles, PSs and BCNs. PSs have been evaluated extensively following multiple routes of administration(13, 16-17, 20). However, the fate of BCNs in vivo has not been reported. We therefore sought to compare BCNs to PSs that we have previously characterized both in vitro and in vivo(13, 16-20). Since PEG17-b-PPS36 PSs and PEG17-b-PPS75 BCNs share the same PEG MW 750 outer corona and a spherical morphology, differences in their intracellular payload delivery and biodistributions can be attributed to their physical differences, such as size and hydrophobic PPS content. PSs and BCNs were successfully fabricated via FNP and subsequently analyzed by DLS, Cryo-TEM and SAXS (Fig. 1). Cryo-TEM analysis confirmed the vesicular morphology of PSs (Fig. 1b) and the presence of internal aqueous channels in BCNs (Fig. 1c). The internal structure of BCNs was also clearly visualized using negative staining TEM analysis (Fig. S1). DLS analysis demonstrated formation of monodisperse populations of PSs and BCNs with number diameters of 72 and 255 nm (Fig. 1d) and polydispersity indices of 0.240 and 0.368, respectively. Both PSs and BCNs morphologies were further confirmed by SAXS analysis. The


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Figure 1. Formation of PSs and BCNs via FNP. (a), diagram of PEG-bl-PPS copolymer, CIJ mixer, and resulting self-assembled nanocarriers. Representative cryoTEM images of (b) PSs and (c) BCNs. Scale bars represent 100 nm. (d) DLS number distribution of diameters for PSs and BCNs. N=3, error bars = S.E.M. (e), SAXS data for PSs and BCNs. PSs were fitted with a vesicular model (SASView) and BCNs were labeled with Braggs peaks demonstrating cubic internal structure.

SAXS scattering profile of PSs was best fitted using a vesicular model with core radius of ~68 nm and shell thickness of ~ 9.5 nm (Fig. 1e). This result agreed with DLS and Cryo-TEM analysis. SAXS scattering profile of BCNs showed Bragg peaks with relative spacing ratios at √2, √4, √6 and indicating the presence of primitive type (Im3m) cubic internal organization (Fig. 1e). This result was consistent with our previous reported SAXS analysis of PEG-bPPS BCNs formed by FNP(12). To summarize, BCNs are larger in diameter than PSs and composed of copolymer chains containing the same hydrophilic (PEG)

mass

but

approximately

twice

the

hydrophobic (PPS) mass. BCNs and PSs have similar spherical morphologies, PEG corona length and surface chemistries, but different internal organization. The larger hydrophobic block length and assembled aggregate diameter of PEG17-b-PPS75 BCNs compared PEG17-b-PPS36 PS (75 propylene sulfide units compared to 36) led us to



hypothesize that BCNs should be capable of loading larger quantities of hydrophobic cargo compared to PSs, on a per-particle basis. To test this, the hydrophobic loading and cellular internalization capacity of BCNs and PSs were compared using ethyl eosin, a model hydrophobic fluorescent dye(14). We have previously loaded ethyl eosin into PSs and BCNs at 0.25 and 0.5 weight % of the polymer mass for demonstrating the capability of these structures to load hydrophobic compounds(12-13). In this case, as we sought to compare the maximum hydrophobic capacity of PSs and BCNs, we utilized a weight percentage of ethyl eosin 4-8 times greater than our previous work: 2 wt % ethyl eosin. Preliminary fluorescence measurements of BCN and PS formulations before and after removal of unencapsulated ethyl eosin suggested that both nanocarriers had similar levels of ethyl eosin encapsulation efficiency at 90-92%. However, macroscopic differences in formulation appearance lead us to suspect that the PS formulations were not forming the typical PS morphology but were rather forming a disrupted morphology due to the overwhelming concentration of ethyl eosin, potentially micellar structures with PEG-b-PPS stabilized cores of ethyl eosin. In order to investigate our suspicions, we performed negative stain TEM on BCNs and PSs that were formed with or without ethyl eosin (Fig. 2a). BCNs formed the same aggregate morphology with or without ethyl eosin loading, characterized by 200-300 nm structures with internal aqueous channels. PSs, however, demonstrated starkly different morphologies when comparing those loaded with ethyl eosin to those without. PSs formed in the absence of ethyl eosin demonstrate vesicular morphology by TEM, in line with observations from cryoTEM (Fig. 1b). PSs failed to form, however, in the presence of 2 wt% ethyl eosin. Rather, small micelle structures were found by TEM (Fig. 2a), suggesting that the high concentration of ethyl eosin was sufficient to disrupt the proper formation of polymersomes, resulting instead in the formation of micelles. These results demonstrate that BCNs are capable of loading higher concentrations of hydrophobic compounds without altering their aggregate morphology in comparison to PSs. To further investigate the comparative hydrophobic volume differences between BCNs and PSs, we sought to adapt a standard assay for determining the critical micelle concentration (CMC) of amphiphiles using the small hydrophobic fluorophore pyrene. The fluorescence emission intensity of pyrene changes depending on the surrounding solvent. At 390 nm, pyrene exhibits reduced emission intensity in aqueous environments and increased emission intensity in hydrophobic environments. This feature is useful for CMC measurements, as small amounts of the amphiphile can be added gradually to aqueous pyrene until micelles form and load the pyrene, increasing the fluorescent signal(22). We reasoned that pyrene in aqueous solution would partition into the hydrophobic domains of assembled nanocarriers over time, and that the


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Figure 2. Comparative Hydrophobic Loading of PSs and BCNs. (a), TEM images of PSs and BCNs with or without 2 wt % ethyl eosin loaded. Arrows and labels help identify example nanocarriers, MC = micelle. Scale bar = 200 nm. (b), fold increase of pyrene fluorescence emission at 390 nm upon excitation at 331 nm in 1xPBS or in the presence of pre-formed BCNs or PSs. Error bars represent S.E.M., n = 3. (c) particle number per µg of polymer for PSs and BCNs, n=3, error bars = S.D., p value determined by Welch’s t test.

process should be quantifiable by detecting an increase in fluorescence intensity. If BCNs do

have increased capacity for hydrophobic compounds compared to PSs, exposing the nanostructures to a pyrene solution should result in a higher fluorescence intensity for the BCNpyrene solution compared to the PS-pyrene solution, and both should outperform pyrene in the absence of added nanocarriers. Indeed, we found that BCNs were able to increase the fluorescence intensity of the pyrene solution by approximately twice as much as PSs (Fig. 2b) after 1 h of mixing. Per the same weight of polymer, PSs outnumber BCNs 1.5:1 (Fig. 2c), a numerical disadvantage that BCNs are able to overcome by loading more pyrene in their extensive hydrophobic volume. 3.2 BCNs achieve high simultaneous dual loading of hydrophilic and hydrophobic cargo and are rapidly internalized by phagocytic cells in vitro One of the benefits of BCNs and PSs over simpler micellar nanocarriers is that they can simultaneously deliver both hydrophilic and hydrophobic molecules into cells. To demonstrate



this advantageous property of these nanocarriers and to compare the loading and uptake between BCNs and PSs, we loaded BCNs and PSs with fluorescein-tagged bovine serum albumin (FITC-BSA), a hydrophilic protein at 2% w/w protein/polymer, and DiD, a hydrophobic fluorescent dye at 0.125% w/w dye/polymer (Fig. 3a). The mass of polymer used was kept the same between PS and BCN formulations at 10 mg per formulation. Due to the structural disruption of PSs we observed with high concentrations of ethyl eosin, we instead chose to load a lower, non-saturating concentration of DiD. Quantification of FITC-BSA and DiD co-loading revealed the BCN encapsulation efficiency to be significantly higher for hydrophilic cargo (67.7% vs 32.6%) and slightly more for hydrophobic cargo (94.6% vs 89.7%) than PSs (Fig. 3b). This agrees well with previous work on BCNs and PSs which also saw improved hydrophilic loading of ovalbumin and dextran, potentially due to the trapping of macromolecular cargo within the water channels of the BCN internal structure(12-13). This result for the similar levels of DiD loading is does not stand in contrast to our earlier demonstration of superior hydrophobic loading in BCNs compared to PSs, as a non-saturating concentration of DiD was used here, and the high hydrophobicity of DiD led to nearly complete loading of the dye into both structures. We assessed the delivery of both the hydrophilic and hydrophobic cargo to RAW 264.7 cells in vitro. RAW 264.7 cells are a murine macrophage cell line that we used here to demonstrate the propensity of phagocytic cells to internalize PSs and BCNs. After incubation with PSs and BCNs for amounts of time ranging from 0.5 to 8 h, RAW 264.7 cells were harvested and analyzed via flow cytometry. FITC-BSA and DiD fluorescence revealed a positive correlation in cell uptake, indicative of co-delivery (Fig. 3c). BCNs and PSs were internalized in increasing amounts over time, as demonstrated by the continually rising median fluorescence intensity (MFI) of the DiD signal in the cells (Fig. 3d). The FITC-BSA signal reaches a maximum at 4 h of incubation (Fig. 3e), a phenomenon best explained by the pH sensitivity of FITC and the acidification of the endolysosomal compartments that carry the BCNs and PSs. The uptake of these nanocarriers is rapid, with >90% of cells positive for BCNs and PSs after just 30 minutes of incubation (Fig. 3f). This increases with time until nearly all cells possess BCN or PS signal. 3.3 BCNs show greater splenic uptake and reduced liver clearance compared to PSs at 4 and 24 h post IV injection.


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Figure 3. PS and BCN Dual Loading and In Vitro Uptake Assay. (a), overview of uptake assay of FITC-BSA DiD dual loaded PSs and BCNs by RAW 264.7 cells. (b), encapsulation efficiency (percentage of cargo successfully loaded out of total initial cargo) of FITC-BSA and DiD in PSs and BCNs when simultaneously loaded, n=3 independent formulations, error bars = S.D., p values from the Holm-Sidak multiple t test. (c), representative contour plots from flow cytometry of RAW 264.7 cells incubated with PSs and BCNs for multiple timepoints. Normalized median fluorescence intensity (MFI) for (d) DiD and (e) FITC-BSA within cells after incubation with PSs and BCNs for increasing amounts of time. (f), percentage of cells that were PS or BCN positive, as determined by gating of the DiD channel. For (d) – (f), n=6, error bars = S.D.



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Nanocarriers injected intravenously (IV) are cleared by the mononuclear phagocyte system (MPS), a multi-organ system consisting primarily of the liver, spleen and kidneys(24). In most cases, the MPS results in a significant majority of the IV administered nanocarriers being taken up by Kupffer cells in the liver(25). In cases where the liver is not the therapeutic target, the MPS therefore serves to reduce the number of circulating nanocarriers capable of reaching the relevant organ or tissue(26). Of the MPS organs, the spleen is of particular interest to those developing immunotherapies, as it is a major immune organ with high levels of B cells, T cells, macrophages, monocytes, and dendritic cells, capable of eliciting strong cellular and humoral immune responses. With increased interest in immunotherapies and cancer vaccines, a nanocarrier system capable of demonstrating reduced liver clearance and increased splenic uptake would be useful for modulating immune responses for therapeutic purposes. We had previously demonstrated that PEG-b-PPS micelles, filomicelles and PSs with the same surface chemistry demonstrated differential uptake in organs(12-13,

17)

. We therefore

sought to elucidate the organ-level biodistribution of BCNs using the same PS from this previous study as a reference. To track the biodistribution of the nanocarriers, we covalently attached maleimide-functionalized DyLight 755, a near-infrared fluorescent dye, to the PEG-bPPS terminal thiol. BCNs and PSs were injected via the tail-vein of C57BL6J female mice, and organs were harvested 4 or 24 h post-injection. The organs were fluorescently imaged and the radiant efficiency of nanocarriers was quantified in each (Fig. 4). Qualitative examination of representative images for the 4 h (Fig. 4a) reveal clear differences in organ-level biodistribution of the two nanocarriers. Representative images for the 4 h and 24 h timepoints are also available in the supplement (Fig. S2), which use an individual color scale to allow for better observation of organ-level distribution of each treatment. BCNs show decreased uptake in the liver (Fig. 4b), and increased uptake in the spleen and lungs (Fig. 4c) compared to PSs. These results suggest that BCNs more effectively avoid liver clearance than PSs, which were previously shown to achieve significantly decreased liver uptake compared to PEG-b-PPS micelles(17). This decreased liver clearance could increase BCN bioavailability in other tissues, such as in the vascular adventitia or the tumor microenvironment. Increased BCN uptake in the spleen suggests it may be an attractive platform for subunit vaccines or other immunomodulatory therapies, as the spleen functions as a major lymphoid organ for immune cell trafficking and signaling(27). The spleen is an important organ for the induction of peripheral tolerance to antigens, which is of increasing interest in cancer immunotherapy and autoimmune disorders(28).


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Figure 4. IVIS Organ-Level Biodistribution of PSs and BCNs. (a) Representative IVIS images for mouse organs harvested 4 h post IV injection of PBS, PSs, or BCNs. Quantification of radiant efficiency of PS and BCN fluorescent signal in the (b) liver and (c) other organs at 4 h and 24 h post IV injection. N = 6 for all organs, 12 points plotted for kidneys representing quantification for the right and left kidneys. Error bars = S.D., significance determined via Sidak’s multiple comparisons test, * p < 0.001, *** p < 0.0001.



Several notable physical and structural differences may contribute to the differences in biodistribution observed between BCNs and PSs. As noted earlier (Fig. 1d), PSs are smaller than BCNs, and size is often considered a major determinant in nanocarrier uptake in the spleen and liver. However, the general trend is for larger particles to be cleared by the liver in greater numbers than smaller particles, which is opposite of the trend found in this study(29). PEG-b-PPS BCNs and PSs have the same surface chemistry, and utilize the same molecular weight PEG, suggesting that their protein corona would be of similar composition. However, BCNs have hydrophilic channels which may increases their accessible surface area for protein adsorption. The relationship between protein adsorption and nanocarrier clearance is complex and depends on the proteins that preferentially adsorb to the surface of the particles, though it is generally thought that increased protein adsorption leads to decreased circulation time and more rapid clearance(30). As such, more detailed examinations of the particular proteins involved in BCN and PS adsorption in vivo will need to be examined in future studies, including work to determine whether the diameter of BCN aqueous channels plays a role in serum protein adsorption levels. PEG-b-PPS PSs appear to demonstrate deformability, which can be observed in cryoTEM images(13), and it is not unreasonable to hypothesize that due to the density of its interior, BCNs might be less deformable. Deformable nanocarriers may be able to squeeze through smaller fenestrations than nanocarriers of the same diameter but of greater stiffness(31). Also, some evidence suggests that nanocarriers better able to deform generate more contacts with cell surfaces, which may change the uptake characteristics of these nanocarriers(32). In short, while several differences exist between PSs and BCNs, more characterization will be required to better understand the relative contributions each variable play in the organ-level biodistribution differences observed here. BCNs demonstrate a transient accumulation in the lungs, which is significantly elevated at 4 h but is largely absent at 24 h post-injection (Fig. 4c). This is likely not due to poor perfusion of the highly vascularized lung tissue, as the BCN fluorescence levels in whole blood at both timepoints is lower than that in the tissue itself (Fig. S2). Long-term accumulation of nanoparticles in the lungs can lead to health concerns if they that cannot be effectively cleared by alveolar macrophages or are inappropriately internalized by local epithelial or mesothelial cells(33). In the case of BCNs in this study, the clearance occurs at a timescale unlikely to cause pathological issue in the lungs. Previously published work comparing the biodistribution of nanoparticles of similar size to the BCNs used in this study found a similar transient accumulation in the lungs, which was not observed with smaller nanoparticles used for lung


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cancer treatment(34). This suggests that the transient accumulation in the lungs may be advantageous for the delivery of drugs to alveolar macrophages, or for the use of BCNs as vaccine vehicles for pulmonary diseases such as tuberculosis. 3.4 BCNs show a similar pattern of uptake by immune cell populations to that of PSs. Many phagocytic immune cell populations exist within the organs in which PSs and BCNs were found to accumulate, and we have previously demonstrated that morphology plays a role in the pattern of uptake of nanocarriers by these cells(17, 19). We performed flow cytometry on cells from the liver, blood, and spleen of mice 4 or 24 h after IV administration of BCNs and PSs (Fig. 5, S3). Previous work using PSs suggested that most uptake in the spleen would be by myeloid cells, so we first sought to compare CD45+ immune cell uptake between PSs and BCNs, excluding lymphoid cells such as B cells, T cells, and NK cells. There was no significant difference in uptake of PSs and BCNs by CD45+ myeloid cells at the 4 h or 24 h timepoints (Fig. 5a,b). However, there is a noticeable increase in nanoparticle positive (NP+) cells in the blood at 24 h, compared to 4 h, for both the PSs and BCNs. This increase is significant for PSs (p < 0.01, Holm-Sidak multiple t-test), but is not significant for BCNs due to increased variability, although the trend appears to be the same. This increase is attributable to an increase in nanoparticlepositive dendritic cells (Fig. 5e,f), although it is important to point out that this is not an increase in dendritic cells as a fraction of immune cells in the blood (Fig. S4a). As mentioned previously, BCNs demonstrated decreased uptake in the liver compared to PSs, based on quantification of IVIS fluorescence (Fig. 4b). As there is no significant difference in uptake between BCNs and PSs by CD45+ cells in the liver, we sought to investigate whether non-immune cells (CD45-) could be responsible for the observed organ level difference in PS and BCN uptake. There was no significant difference between PS and BCN association at 4 h (Fig. 5c) but there was a significant decrease in association of BCNs with CD45- cells at 24 h compared to PSs (Fig. 5d). This trend in the liver exists at the 4 h timepoint but was not significant. CD45- cells make up a considerable majority of the total number of cells within the liver, such as hepatocytes and liver endothelial cells(35). We do note that the cell processing methodology adopted for flow cytometry of the liver for this paper may result in the underrepresentation of some CD45- cells, particularly hepatocytes. To summarize, both nanocarriers were internalized by myeloid cells in the blood, spleen, and liver, many of which function as professional antigen presenting cells that are key targets during immunotherapy and



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Figure 5. Flow Cytometric Assessment of Cell Population Uptake of PSs and BCNs After IV Injection. Nanoparticle uptake of PSs + and BCNs by CD45 myeloid cells at (a) 4 h and (b) 24 h post IV injection and by CD45 cells at (c) 4 h and (d) 24 h post IV injection. + Nanoparticle positive (NP ) myeloid cells were identified + as CD45 CD3 CD19 NK1.1 Ly6G . Dendritic cell (DCs) uptake of (e) PSs and (f) BCNs, comparison of percentage of DCs that were nanoparticle positive at 4 hours vs 24 hours post injection. Dendritic cells were + identified as CD45 CD3 CD19 NK1.1 Ly6G F4/80 + CD11c . For all graphs, significance determined using Holm-Sidak multiple t-tests, p values shown in graph. Error bars = S.E.M., n = 3.


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vaccination. The pattern of uptake of the two nanoparticles by immune cells is largely the same, with no significant differences when broken down by subpopulations of immune cells (Fig. S3). In the spleen, where a bulk of the cells are of the lymphoid lineage (B cells, T cells) that do not take up a large number of nanoparticles, it can be informative to indicate which populations were most represented out of the NP+ cells. In Figure 6, each circle represents all the cells that were NP+ in the spleen, split into slices that represent distinct cell populations. Despite being a small minority of the cells within the spleen (

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