Dense and Dynamic Polyethylene Glycol Shells Cloak Nanoparticles

DiD labeling is well-accepted in NP circulation studies because of the slow release of DiD from NPs,(33) as confirmed by us that around 7% of DiD was ...
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Dense and Dynamic Polyethylene Glycol Shells Cloak Nanoparticles from Uptake by Liver Endothelial Cells for Long Blood Circulation Hao Zhou,† Zhiyuan Fan,† Peter Y. Li,† Junjie Deng,†,‡ Dimitrios C. Arhontoulis,∥ Christopher Y. Li,† Wilbur B. Bowne,§ and Hao Cheng*,†,∥ †

Department of Materials Science and Engineering, Drexel University, Philadelphia, Pennsylvania 19104, United States Engineering Research Center of Clinical Functional Materials and Diagnosis and Treatment Devices of Zhejiang Province, Wenzhou Institute of Biomaterials and Engineering, CAS, Wenzhou 325011, China ∥ School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104, United States § Department of Surgery, Drexel University, Philadelphia, Pennsylvania 19102, United States ‡

S Supporting Information *

ABSTRACT: Research into long-circulating nanoparticles has in the past focused on reducing their clearance by macrophages. By engineering a hierarchical polyethylene glycol (PEG) structure on nanoparticle surfaces, we revealed an alternative mechanism to enhance nanoparticle blood circulation. The conjugation of a second PEG layer at a density close to but lower than the mushroom-to-brush transition regime on conventional PEGylated nanoparticles dramatically prolongs their blood circulation via reduced nanoparticle uptake by non-Kupffer cells in the liver, especially liver sinusoidal endothelial cells. Our study also disclosed that the dynamic outer PEG layer reduces protein binding affinity to nanoparticles, although not the total number of adsorbed proteins. These effects of the outer PEG layer diminish in the higher density regime. Therefore, our results suggest that the dynamic topographical structure of nanoparticles is an important factor in governing their fate in vivo. Taken together, this study advances our understanding of nanoparticle blood circulation and provides a facile approach for generating long circulating nanoparticles. KEYWORDS: protein corona, nanomedicine, drug delivery, complement activation, grafted polymer, internalization

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attributed to the increased PEG density and layer thickness because short PEG chains fit in between longer PEG molecules, and steric repulsions between grafted chains increase the overall thickness of the PEG layer. However, some studies have reported the existence of an optimal density of grafted PEG for repelling proteins.20−22 One explanation from theoretical analysis is that the PEG layer transitions from a mushroom regime to a brush regime as the polymer grafting density increases.23 A mushroom regime refers to a low PEG density, in which the dynamic chain coils rarely interact laterally on surfaces, while a brush regime is a higher density condition where chains are confined and extended from

anoparticle (NP)-based therapeutics and imaging agents are promising for many applications. However, successful translation of synthetic NPs to human use has been limited by their short circulation half-lives in the bloodstream. This clearance is believed to be primarily due to opsonin adsorption on NP surfaces, making them visible to cells of the mononuclear phagocyte system.1−3 PEG and other polymer grafts on NPs are widely used to reduce protein adsorption, complement activation, and prolong NP circulation.2−12 It is generally accepted that a thick and dense PEG layer is necessary for this purpose as dense chains prevent protein adsorption by steric repulsion.13−16 Furthermore, polydisperse PEG brushes of varying length have been shown to be more effective than monodisperse PEG layers in reducing protein adsorption,17,18 indicating the design may generate long circulating NPs.19 The improvement was © XXXX American Chemical Society

Received: June 30, 2018 Accepted: August 17, 2018 Published: August 17, 2018 A

DOI: 10.1021/acsnano.8b04947 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. Long blood circulation half-lives of PLGA-PEG-NPs with a dynamic topographical structure of PEG shell (PLGA-TPEG-NPs). (a) Schematic illustration of PLGA-TPEG-NP preparation. PLGA-TPEG-NP-20 indicates that the NPs were prepared with 20% of PLGA-PEGMAL and 80% of PLGA-PEG and further modified with methoxy PEG2K-thiol to generate a hierarchical shell structure. (b) A representative TEM image of PLGA-TPEG-NPs-20 (scale bar: 100 nm). (c) Blood circulation of PLGA-TPEG-NPs measured by fluorescence intensity of NPs in the plasma. The NPs were prepared with 0%, 10%, 20%, 40%, 60%, 80%, and 100% PLGA-PEG-MAL. Values indicate mean ± SD (n = 6, from two independent experiments). All NPs were fluorescently labeled by encapsulating DiD. (d) Circulation half-lives of PLGATPEG-NPs obtained by fitting the circulation profile data (c) into a one-compartment model via PKSolver. Values indicate mean ± SD (n = 6). *, P < 0.05, ***, P < 0.001. (e) Schematic illustration of the proposed three-dimensional PEG structure on NP surfaces with an increased PEG density in the outer PEG layer. The primary layer of PEG forms a dense polymer brush, which repels proteins sterically. The outer layer of PEG changes from mushroom to brush regime as PLGA-PEG-MAL percentage increases. Within each blob, the PEG chain behaves as a free polymer in a good solvent. (f) Calculated average distance of neighboring chains (D) and thickness (L) of the outer PEG layer, which determines the topographical structure of PEG shells. The calculation followed de Gennes’ model of grafted polymers on a flat surface in a good solvent. To simplify the estimation, L ≈ Na5/3D−2/3 was applied when D < RF. Values indicate mean ± SD (n = 3). Two methods were used to calculate D and L: (1) a standard method using hydrated NP size from DLS to calculate NP concentration and surface area; and (2) a method using dehydrated NP size from TEM to calculate NP concentration.

fluctuation effect to kinetically interfere with protein binding, leading to long NP circulation. Although NP physicochemical properties such as shape, size, elasticity, surface chemical composition, crystallization, and stability have been investigated for extending NP circulation,15,27−32 the impact of NP dynamic topographical structures has yet to be elucidated. Indeed, we found that an outer layer of PEG at a density close to the mushroom-to-brush transition regime creates an ideal topographical structure to enhance blood circulation and tumor accumulation of conventional PEGylated NPs. Surprisingly, the dramatically extended NP circulation time stems from reduced NP uptake by liver sinusoidal endothelial cells (LSECs) rather than macrophages, which have been the focus of previous studies of generating long circulating NPs.2,15,33−35 In addition to providing a different mechanism for generating long circulating NPs and revealing the dynamic topographical

substrates.24 The thermally agitated conformational fluctuation of low-density PEG chains increases the characteristic adsorption time and therefore kinetically slows down protein adsorption, while confined chains at a high PEG grafting density do not have this effect.23 Therefore, there is a fundamental design trade-off between steric repulsion and conformational fluctuation. Understanding and maximizing both effects would substantially advance polymer graft design for enhanced NP circulation. Here, we report the generation of a PEG cloak with dual physical effects for NPs (Figure 1a). This design was based on the hypothesis that NPs with a dense inner (primary) PEG layer would thermodynamically prevent proteins from accessing NPs via steric repulsion,25,26 while an outer layer of PEG in the mushroom regime would provide a topographical structure for NPs for harnessing the conformational B

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convenient to functionalize NPs with biomolecules on the primary PEG layer instead of the outermost layer to minimize the conjugated biomolecule-induced NP clearance.38 It is known that a fraction of PEG chains may entrap inside NP hydrophobic cores such as PLGA during nanoprecipitation.39 The two-step NP fabrication allows us to quantify PEG chains in the outer PEG layer for determining their conformation. Outer PEG Layer Close to the Mushroom-to-Brush Transition Regime Is Optimal for NP Circulation. To understand the topographical chain conformation that affects NP blood circulation, the chain densities in the outer PEG layer were evaluated using FITC-PEG2K-thiol in the conjugation step and measuring the FITC fluorescence intensity of NP solutions.40 The chain densities can be obtained after knowing FITC-PEG2K concentration, NP concentration, and NP size. The distance between neighboring chains (D) was calculated using

structure as a factor for consideration in NP design, this study helps to fundamentally understand some previously reported results of PEGylated NPs.

RESULTS AND DISCUSSION Dynamic Topographical Structure of PEG Shells Prolongs the Circulation of PEGylated NPs. Widely used poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) NPs (PLGA-PEG-NPs) were first selected for this study. To create PLGA-PEG-NPs with controlled topographical structures of PEG shell (PLGA-TPEG-NPs), methoxy PLGA20K-PEG5K was mixed with maleimide-terminated PLGA20K-PEG5K (PLGAPEG-MAL) and formed maleimide-functionalized PLGA-PEGNPs via nanoprecipitation.36 The resulting particles were then coupled with methoxy-PEG2K-thiol via the thiol-maleimide reaction to generate a hierarchical PEG shell (Figure 1a). The polydispersity index of PEG molecules or blocks was approximately 1.05. To study the effect of topographical structure on NP blood circulation, PLGA-TPEG-NPs with a series of PLGA-PEG-MAL percentages were prepared (PLGATPEG-NPs-10, 20, 40, 60, 80, and 100, in which the numbers indicate the percentages of PLGA-PEG-MAL in NP fabrication). NPs with different outer layer PEG densities were fabricated to have similar average sizes between 85 and 97 nm and similar ζ potentials at −2 mV (Figure S1). The average size of PLGA-TPEG-NPs-20 measured with dynamic light scattering (DLS) was approximately 90 nm (Figure S1b), while it was around 50 nm (48.6 ± 8.9 (SD) nm) in the transmission electron microscopy (TEM) image of the NPs (Figure 1b) similar to those of PLGA-PEG-NPs and PLGA-TPEG-NPs100 (Figure S2). Further characterization showed that the remaining maleimide on the NP surfaces was undetectable (99%) during the conjugation step (Figure S3). The circulation of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiD)-labeled NPs in the blood was evaluated in BALB/c mice based on the fluorescence signal of DiD in the plasma (Figure 1c). DiD labeling is well-accepted in NP circulation studies because of the slow release of DiD from NPs,33 as confirmed by us that around 7% of DiD was released from NPs in vitro after 48 h (Figure S4). The circulation data were analyzed to calculate circulation half-lives (Figure 1d) and other pharmacokinetic parameters (Table S1) by applying a pharmacokinetic one-compartment model via PKSolver.37 PLGA-TPEG-NPs-20 showed the longest circulating half-life of 10.01 ± 0.52 h, which was approximately three times the 3.48 ± 0.36 h circulation half-life of conventional PLGA-PEGNPs made of 100% methoxy-PLGA20K-PEG5K. The circulation half-life of NPs started to decrease when the density of the outer PEG layer further increased. PLGA-TPEG-NPs made of 80% and 100% PLGA-PEG-MAL showed similar circulation half-lives to the conventional PLGA-PEG-NPs, even though they possess longer elimination half-lives when calculated using a two-compartment model (Table S2). To exclude the effect of the maleimide-thiol linkage on NP circulation, methoxy-PEG-thiol with two ethylene glycol units was used to quench the maleimide groups. The resulting PLGA-PEG-NP-20 has the same amount of maleimide-thiol linkage as PLGA-TPEG-NP-20, but without the outer PEG layer. Its blood circulation profile was similar to that of conventional PLGA-PEG-NPs (Figure S5), indicating the prolonged circulation of PLGA-TPEG-NP-20 results from the outer PEG layer. This two-step NP fabrication method is

D ≈ A1/2 in which A is the average area occupied by one PEG chain. The Flory radius (end-to-end distance) of an unperturbed PEG coil in the outer layer is calculated using24,41 RF ≈ N3/5a

in which N is the degree of polymerization (N = 45 for PEG2K), and a is the monomer size, (a = 0.35 nm for PEG).40 In the outer layer, the thickness in the mushroom regime (D > RF) and mushroom-to-brush transition regime is simply equal to or close to RF, while in the brush regime (D ≪ RF), the thickness can be estimated using24 L ≈ Na5/3D−2/3

Experimental study of grafted PEG2K has shown that the layer thickness in the mushroom regime is RF, matching well with the theoretical estimation. It was also found that the PEG layer thickness started to increase when D was reduced to around RF (A ≈ RF2),42,43 reaching the standard mushroom-tobrush transition regime.24,41 The hydrated NP sizes were first used in the estimation of NP concentrations, surface areas, and PEG densities in the outer layers.44,45 This standard method in the field showed the maximum circulation time occurs when the outer layer PEG density is at D ≈ RF (Figure 1f). However, the standard method underestimated NP concentrations in solutions because hydrated NPs include the weight of water molecules. When the dehydrated NP size from TEM (50 nm) was used to calculate NP concentrations and hydrated NP sizes were used to calculate surface areas, the optimal PEG density in outer layer was at D ≈ 2RF, in which the mushrooms are close to each other, but have no appreciable lateral interactions. The optimal outer layer PEG density is reasonable to be slightly lower than the mushroom-to-brush transition regime. This is because the lateral interaction among neighboring PEG chains at D ≈ RF can significantly reduce chain compressibility,42 limiting chain fluctuation. Considering the diameters of plasma proteins are mostly around or larger than RF (3.5 nm), PEG chains at a density D ≈ 2RF, which allows free chain fluctuation, may be sufficient to protect NP surfaces via interfering with protein binding. Dynamic Outer PEG Layer Reduces Protein Binding Affinity on PEGylated NPs. One of the major factors in determining NP fate in vivo is their protein corona, which is formed via protein adsorption on NPs in the blood.46,47 To C

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Figure 2. Isothermal titration calorimetry (ITC) study of protein binding on NPs. The dynamic topographical structure of PEG shell reduces the affinity of protein binding on NPs. (a−d) Representative ITC data. Graphs show integrated heat of each titration (■) with a corresponding fitted curve based on the one-site binding model for (a) PLGA-NPs, (b) PLGA-PEG-NPs, (c) PLGA-TPEG-NP-20, and (d) PLGA-TPEG-NP-100. (e,f) Calculated stoichiometry (Na) and binding affinity (Ka) for the indicated samples. Values indicate mean ± SD (n = 3). ***, P < 0.001.

chances to interact with fluctuating PEG chains than their smaller sized counterparts. Dynamic Outer PEG Layer Reduces the Clearance of PEGylated NPs by Non-Kupffer Cells in the Liver. To understand how the dynamic outer PEG layer affects NP clearance in the blood circulation, NP uptake by cells was studied in vivo. The 4 h time point after systemic NP administration was selected for the study because there were approximately 60% of conventional PLGA-PEG-NPs and 30% of PLGA-TPEG-NPs-20 cleared at the time (Figure 1c), giving a significant difference for comparison. According to the IVIS image of tissues collected at this time point (Figure S7), NPs were mainly cleared by the liver and spleen. PLGA-TPEGNPs-20 showed a similar biodistribution as conventional PLGA-PEG-NPs in most tissues except a significantly lower uptake in the livers. Therefore, the in vivo study of NP cellular uptake was focused on liver cells. For the study, the livers were perfused with a collagenase solution in situ, and the isolated cells were analyzed with flow cytometry. It was found in the histograms of total isolated cells that liver cells from the mice treated with PLGA-PEG-NPs showed a single peak (A) at a position of high DiD (NP fluorescence) intensity, while liver cells from PLGA-TPEGNP-20-treated mice showed two peaks at positions of intermediate and high DiD intensities (B and C) (Figure 3a). The peak split and shift demonstrate that the dynamic outer PEG layer reduces the uptake of PEGylated NPs in some liver cells. To identify the cells, the isolated cells were classified into four groups according to marker staining and forward and side scatter of light: hepatocytes (large CD146−CD68− cells), LSECs (CD146+CD68− cells), Kupffer cells (CD146−CD68+ cells), and other cells (small CD146−CD68− cells) including hepatic stellate cells, B cells, and others (Figure S8). Hepatocytes, LSECs, and Kupffer cells are the most abundant cells in livers and have been reported to account for 57%, 23%, and 15% of total liver cells, respectively.50 The analysis of DiD+ cells shows that the majority of LSECs and Kupffer cells

understand whether the outer PEG layer at the optimal density that gives the longest circulating NPs affect protein adsorption, NP−protein interactions were analyzed using isothermal titration calorimetry (ITC), which can directly measure protein adsorption to NPs in physiological media.48 Saline with 20% fetal bovine serum (FBS) was titrated into the NP solutions, and the change in heat for every titration was measured (Figure S6) and integrated (Figure 2a−d). The resulting integrated heat was fitted into a one-site binding model where the protein adsorption enthalpy (ΔH), stoichiometry (Na, which is the average number of proteins adsorbed on a single NP), and binding constant (Ka) were calculated (Figure 2e,f).40 Since whole serum proteins exist in the system, the detected Na and Ka are apparent numbers, reflecting the “average” interaction between NPs and proteins. The adsorption of serum proteins on the surfaces of PLGANP, PLGA-PEG-NPs, PLGA-TPEG-NPs-20, and PLGATPEG-NPs-100 is all exothermic. The results from PLGANPs and PLGA-PEG-NPs are in agreement with previous studies that PEGylation reduces protein adsorption.40,44 Interestingly, PLGA-PEG-NPs, PLGA-TPEG-NPs-20, and PLGA-TPEG-NPs-100 showed similar numbers of adsorbed proteins on NPs (Na). This is likely because the NPs have a similar particle size and PEG density in the primary PEG layer, resulting in similar numbers of protein binding sites. Some studies have shown that PEG density is the most important factor in determining the amount of adsorbed proteins on substrates, while variation of PEG length has a limited effect.49 PLGA-TPEG-NPs-100 can be simply considered as PLGAPEG-NPs with a longer PEG length. However, the binding affinity of proteins to NPs with a hierarchical PEG shell (PLGA-TPEG-NPs-20) was significantly less than those of the conventional PLGA-PEG-NPs and PLGA-TPEG-NP-100. This dynamic effect of the outer PEG layer may affect the adsorption of proteins that mediate NP clearance directly or indirectly as proteins interact with those that are already adsorbed on NPs. This dynamic effect is also expected to be more dramatic for large sized proteins because they have more D

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Figure 3. Dynamic topographical structure of PEG shells significantly reduced the uptake of PEGylated NPs by endothelial cells in the liver. Liver cells are stained with CD146 and CD68. (a) Representative histogram of NP internalization of all cells in the livers collected from mice 4 h after tail vein injection of saline, PLGA-PEG-NP, or PLGA-TPEG-NP-20. (b) Percentage of cells that internalized NPs (DiD+) within hepatocytes, endothelial cells, Kupffer cells, and other cells. (c−f) Representative histograms of NP internalization of endothelial cells, Kupffer cells, hepatocytes, and other cells in the livers. (g) Relative mean fluorescence intensity (MFI) of different liver cells. (h) Relative MFI of hepatocytes that internalized NPs or not (NP+/NP−). (i) Relative MFI of other cells that internalized NPs or not (NP+/NP−). Values indicate mean ± SD (n = 5 from three independent experiments). *, P < 0.05, ***, P < 0.001.

scavengers of NPs in the blood.2,15,33−35 Although LSECs express scavenger receptors, have a high endocytic capacity beyond the complement-mediated internalization,51 and can uptake NPs,51−54 it was largely unknown that reducing NP internalization by LSECs, but not macrophages, can be sufficient to dramatically extend NP blood circulation. The slight increase of PLGA-TPEG-NPs-20 in Kupffer cells may be because more PLGA-TPEG-NPs-20 than PLGA-PEG-NPs are locally available to Kupffer cells, which spread on top of LSECs in sinusoids, when LSECs are not efficient in removing PLGATPEG-NPs-20. There were