Role of Liposome Size, Surface Charge, and PEGylation on

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Role of liposomes size, surface charge and PEGylation on rheumatoid arthritis targeting therapy Hongwei Ren, Yuwei He, Jianming Liang, Zhekang Cheng, Meng Zhang, Ying Zhu, Chao Hong, Jing Qin, Xinchun Xu, and Jianxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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ACS Applied Materials & Interfaces

Role of liposomes size, surface charge, and PEGylation on rheumatoid arthritis targeting therapy Hongwei Ren a, Yuwei He a, Jianming Liang a, Zhekang Cheng b, Meng Zhang c, Ying Zhu d, Chao Hong a, Jing Qin a, Xinchun Xu e *, Jianxin Wang a, f *

a

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug

Delivery, Ministry of Education, Shanghai 201203, China b School

of Pharmacy, Minzu University of China, Beijing 100081, China

c Shanghai d

University of Traditional Chinese Medicine, Shanghai 201203, China

Institute of Clinical Pharmacology, Guangzhou University of Traditional Chinese Medicine, Guangzhou

510006, China e Shanghai f

Xuhui Central Hospital, Shanghai 200031, PR China.

Institute of Integrated Chinese and Western Medicine, Fudan University, Shanghai 200040, China

Corresponding author: Xinchun Xu Shanghai Xuhui Central Hospital, 966 Middle Huaihai Road, Shanghai 200031, PR China. Tel: 86-21-31270810; Fax: 86-21-31270810; Email: [email protected] Jianxin Wang School of Pharmacy, Fudan University; Key Laboratory of Smart Drug Delivery, Ministry of Education, 826 Zhangheng Road, Shanghai, 201203, China Tel.: +86-21-51980088; fax: +86-21-51980088; E-mail address: [email protected]

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KEYWORDS: liposomes, rheumatoid arthritis, size, surface charge, PEG, targeting, retention

ABSTRACT Rheumatoid arthritis (RA) is a chronic, systemic, progressive autoimmune disease. The vascular permeability of inflamed joints in RA makes it a natural candidate for passive targeting, similar to the enhanced permeability and retention (EPR) effect in solid tumors. Thus, various therapeutic drugs have been encapsulated in nanocarriers to achieve longer in vivo circulation times and improve RA targeting. Although liposomes are the most widely used nanocarriers for RA treatment, the effects of physical and chemical characteristics of liposomes, such as particle sizes, surface charge, polyethylene glycol (PEG) chain length, and PEG concentration, on their passive RA targeting effect have not been fully elucidated. Here, we systematically investigated the effects of physical and chemical properties of liposomes on circulation time, and conducted preliminary studies on their passive targeting mechanisms. Methods: A series of liposomes with different particle sizes (70 nm, 100 nm, 200 nm, and 350 nm), surface charges (positive, negative, slight positive, and slight negative), and PEG chain lengths (1 kDa, 2 kDa, and 5 kDa) and concentrations (5%, 10%, and 20% w/w of total lipid) were prepared by lipid film dispersion and extrusion. The pharmacokinetics of liposomes with different formulas were evaluated with a fluorescence microplate reader. A collagen-induced arthritis (CIA) mouse model was utilized to mimic RA pathological conditions, and to evaluate the targeting and efficacy of liposomes with different properties using a nearinfrared fluorescence (NIRF) imaging system. Uptake of fluorescent liposomes by various synovial cells was measured by flow cytometry. Results: These results indicated that liposomes with 100 nm diameter, slight negative charge, and 10% incorporation of 5 kDa PEG had better in vivo circulation time and inflamed joint targeting than other liposomes had. Dexamethasone (Dex) was encapsulated into optimized liposomes as an active ingredient for ACS Paragon Plus Environment

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RA treatment. Pharmacodynamic studies demonstrated that Dex liposomes could significantly improve the anti-arthritic efficacy of Dex in a CIA mouse model of RA. This study also found that the retention mechanism of RA was mainly increased due to the uptake of liposomes by fibroblasts and macrophages in inflamed joints. Conclusion: This study provides a persuasive explanation for passive RA targeting by liposomes, and advances our ability to treat RA with nanomedicine. INTRODUCTION Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by acute synovitis, progressive articular cartilage erosion, and bone destruction1. The pathogenesis of RA presents symmetrically in multiple joints, and can cause severe disability2. Pharmacological treatments for RA can be categorized into four classes: disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GC), and immune-modulators3. Almost all current conventional drugs require high and frequent dosing due to the inability to target inflamed joints. Poor bioavailability, high clearance rates, and high-dose related toxicities of these drugs seriously restrict their therapeutic effect in RA4. Although intraarticular administration may avoid high dose-related systemic side effects, patient compliance is poor due to the severe pain caused by repetitive intra-joint injections5-6. The emergence of nanomedicine provides a new approach to treat RA. Various novel carriers such as liposomes7, polymer nanoparticles8, albumin nanoparticles9, membrane nanoparticles10, dendrimers11, and gold nanoparticles12 have been applied to the treatment of RA. Among them, liposomes, the most extensively investigated carriers, due to their low immunogenicity, high biocompatibility, and the feasibility of controlling their physicochemical properties and surface modifications13. They can improve the stability and prolong the biological half-life of drugs, and deliver a medicine specifically to inflamed joints14. Their targeting ability is mainly attributed to the leaky vasculature of the synovium caused by inflammatory cytokines, resembling the EPR effect in solid tumors15. In fact, leaky vessels occur in a variety of tumors16 and inflammatory diseases17. ACS Paragon Plus Environment


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The size of the gaps between endothelial cells is different from one disease to another, but fall within a certain range18-19. The physicochemical properties of nanocarriers play a critical role in utilization of the passive targeting feature offered by leaky vasculature at diseased sites. Thus, these relationships should be clearly elucidated. The passive targeting capacity of liposomes is mainly dependent on their enhancement of blood circulation time, which is affected by several parameters, including vesicle size, surface charge, and surface hydrophobicity20. Particle size is the most crucial characteristic of liposomes, which affects their circulation time and biodistribution after intravenous administration21. For example, 450–500 nm liposomes have higher reticuloendothelial system (RES) mediated blood clearance rates than 90–100 nm liposomes22. However, extremely small particles, for example, quantum dots less than 5.5 nm, will easily penetrate the kidney’s filtering system and be rapidly eliminated23. Liposome size also influences their infiltration through gaps in leaky synovial vessels and retention in inflammatory joints. A suitable liposome particle size does not necessarily result in optimally targeted RA therapy. The surface charge of liposomes also significantly influences its behaviors in vivo, including circulation time24. Conflicting results have been reported on the effect of nanocarrier surface charge on circulation time25-26. Therefore, the optimum surface charge for RA-targeting liposomes has not yet been established. Due to adsorption to plasma proteins, and recognition and phagocytosis by the RES system, hydrophilic modifications of liposomes can prolong their blood circulation time27-28. The most widely used hydrophilic polymer is polyethylene glycol (PEG). Research has indicated that PEGylated liposomes could effectively reduce adsorption to plasma proteins (opsonization) and substantially prolong circulation time relative to nonPEGylated liposomes29. The efficacy of preventing opsonization by PEGylation of liposomes is mainly determined by the chain length and concentration of PEG 30-31. To our knowledge, the effects of particle size, surface charge, and PEGylation on RA targeting by liposomes have not been previously reported, and are key to the efficacy of this novel drug delivery system in RA ACS Paragon Plus Environment

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treatment. In this study, we systematically evaluate the role of particle size (70–350 nm diameter spheres), surface charge (positive, slightly positive, slightly negative and negative), PEG chain lengths (1 kDa, 2 kDa, 5 kDa, and 10 kDa), and PEG concentration

(5%, 10% and 20% w/w of total lipid) on blood circulation time

and on accumulation in RA inflamed joints (Figure 1A). To evaluate the pharmacodynamics of drug delivery by liposomes, dexamethasone (Dex) was chosen as an RA therapeutic, encapsulated into liposomes, and injected into collagen-induced arthritis (CIA) mice. Arthrotropism and joint retention are two key aspects of RA-targeting therapy. The inflamed joint-tropism of nanocarriers is based on their penetration of the highly permeable synovial vasculature, similar to the EPR effect in tumors32. As already known, the retention mechanism of nanocarriers in tumors is caused by a poorly developed lymphatic drainage system33. In contrast, lymphatic drainage in RA is normal or even accelerated34. Therefore, the possible mechanisms that could contribute to retention of liposomes is yet unknown. Activated synoviocytes, rather than unactivated cells, have been reported to have stronger phagocytic capacity during inflammatory conditions, which might contribute to liposome retention in RA35-38. Similar to the EPR effect in tumors, this phenomenon is known as the “ELVIS” (extravasation through leaky vasculature and subsequent Inflammatory cell-mediated sequestration) effect39. However, the types of cells playing major roles in this retention mechanism have not been systematically elucidated. In this study, fluorescence-activated cell sorting (FACS) was used to investigate the ability of synovium-derived macrophages, fibroblasts, and endothelial cells to phagocytose liposomes (Figure 1B).



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Figure 1. (A) Liposome characteristics that influence systemic delivery and passive targeting of RA. Liposomes can be prepared with different sizes (70–350 nm) and surface charges (positive charge, negative charge, slight positive charge, and slight negative charge). Liposomes can be coated with varying amounts (5%, 10% and 20% w/w of total lipid) of PEG of different chain lengths (1 kDa, 2 kDa, and 5 kDa). (B) Schematic of the RA targeting effect of liposomes in the inflammatory microenvironment of RA. Liposomes were mainly taken up by macrophages and fibroblasts after entering the joint cavity through the leaky vasculature. Fewer liposomes were taken up by endothelial cells. RESULTS AND DISCUSSION Characterization of liposomes Liposomes were quantitated based on their particle sizes and zeta potential by dynamic light scattering at 25 °C. Physical characteristics of liposomes with various sizes, surface charges, PEG lengths, and PEG concentrations are listed in Table 1. Images of liposome morphology were obtained by transmission electron microscopy (Figure 2). It is noteworthy that the size of liposomes was reduced to 350 nm with a narrow PDI by passage through a polycarbonate membrane of 400 nm. Conversely, because of their fluidity, 70 nm liposomes were obtained by passage through a 50 nm polycarbonate membrane40. As shown in Table 1, liposomes of various sizes and surface charges were successfully prepared. PEGylation of liposomes had no significant influence on particle size, meanwhile zeta potential was slightly decreased when chain length of PEG reached 5 kDa. Alteration of PEG5000 content had little effect on the surface charge of liposomes (Table 1). Dexamethasone was encapsulated into liposomes with an efficiency of 62.66 ± 1.81%, and 1.71 ± 0.05% drug loading. Table 1. Particle size, PDI, and Zeta potential of various liposomes Particles

Size (nm)

PDI

Zeta potential (mV)

70 nm

68.18 ± 2.64

0.038 ± 0.017

-30.3 ± 1.75

100 nm

93.94 ± 1.18

0.066 ± 0.009

-33.8 ± 1.05


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200 nm

202.93 ± 5.42

0.225 ± 0.012

-36.5 ± 1.01

350 nm

345.73 ± 12.35

0.167 ± 0.052

-36.5 ± 0.69

Negative charge

92.04 ± 0.78

0.14 ± 0.006

-31.93 ± 1.04

Slight Negative charge

92.6 ± 0.62

0.073 ± 0.022

-15.07 ± 0.85

Slight Positive charge

91.57 ± 0.82

0.139 ± 0.02

7.89 ± 0.94

Positive charge

87.19 ± 2.6

0.094 ± 0.023

37.83 ± 1.64

PEG 1000

96.48 ± 0.42

0.076 ± 0.005

-39.9 ± 0.92

PEG 2000

98.74 ± 1.41

0.068 ± 0.006

-41.93 ± 0.4

PEG 5000

104.8 ± 1.31

0.063 ± 0.02

-28.67 ± 0.51

PEG 5000-5%(w/w)

93.52 ± 1.65

0.081 ± 0.017

-24.93 ± 0.06

PEG 5000-10%(w/w)

93.07 ± 0.65

0.058 ± 0.025

-26.3 ± 0.53

PEG 5000-20%(w/w)

94.68 ± 2.49

0.089 ± 0.02

-21.97 ± 0.5

Figure 2. TEM images of liposomes of different average sizes. A) 70 nm; B) 100 nm; C) 200 nm; and D) 350 nm. Scale bar, 100 nm. Effects of particle size of liposomes on pharmacokinetics, in vivo targeting efficacy and biodistribution DiD loaded liposomes in a range of particle sizes (70 nm, 100 nm, 200 nm, and 350 nm) were injected into healthy mice to investigate their pharmacokinetic profiles. Figure 3A indicates that 100 nm liposomes had a longer blood circulation time than the other three sizes. Overall, 70 nm and 100 nm liposomes had similar pharmacokinetic parameters, which were better than those of the 200 nm and 350 nm liposomes. In this study, ACS Paragon Plus Environment


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the larger liposome sizes had shorter T1/2 and MRT0→t, and lower AUC0→t (Figure 3B). The shorter circulation time of 200 nm and 350 nm liposomes might be due to their large size, which was easily recognized and phagocytized by the RES system41-42. The rapid clearance of small liposomes could be attributed to uptake by the liver43 and filtration by the kidneys44. To evaluate the effect of size on targeting of inflamed joints, DiD-labeled liposomes of various sizes were administrated intravenously to CIA model mice (n = 3). The fluorescence intensities of inflamed paws were determined by in vivo fluorescence imaging. Live imaging of the 100 nm liposome group showed fluorescence intensities in the hind paws that were much higher than those of the other three groups. Fluorescence intensities in this group reached a peak at 12 h post-injection (Figure 3C). The relative fluorescence intensities in mice treated with 100 nm liposomes (n = 3; mean ± SD) were stronger than those in the other groups at each time point surveyed in each region of interest (ROI) (Figure 3D). In addition, the targeting index of the inflamed joints at 48 h post-injection showed that the targeting effect decreased with increasing particle size (Figure 3E). The gaps in synovial endothelial vascular cells caused by inflammation are in submicron dimensions45, which would not hinder liposomes of any tested size from infiltrating gaps in leaky synovial vessels. Therefore, longer circulation times could result in higher accumulation of liposomes in inflamed joints and improve the in vivo targeting efficiency of 100 nm liposomes. The biodistribution of liposomes of different sizes was estimated by fluorescent images of the organs and inflamed paws obtained at 48 h post-injection. These images suggested that the fluorescence signal from the inflamed paws of mice treated with 100 nm liposomes was the strongest among all groups (Figure 3F), which confirmed that the smallest and largest liposomes were cleared most rapidly by the liver, kidneys, and spleen. Distribution in the kidney was the lowest for the 70 nm liposomes, suggesting that the small particle size liposomes were most efficiently filtered by the glomeruli46. Based on the pharmacokinetics and biodistribution results, the 70 and 100 nm liposomes have the best circulatory retention times, significantly better than the 200 and 350 nm liposome groups, and relatively ACS Paragon Plus Environment

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similar to each other. The somewhat higher clearance rate of the 70 nm liposomes relative to 100 nm liposomes is mainly due to the latter’s lower relative renal clearance rate. Thus, 100 nm is the optimal particle size for liposomes for best RA targeting.

Figure 3. Pharmacokinetics, in vivo targeting, and biodistribution of different sized liposomes. (A-B) Pharmacokinetic profiles of various sizes of liposomes in healthy mice (mean ± SD, n = 3). AUC, area under the fluorescence intensity-time curve. T1/2, half-life. MRT, mean residence time. (C) In vivo fluorescence ACS Paragon Plus Environment


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imaging of inflamed paws (AI score>3) (left and right, A) in CIA model mice after intravenous injection of different sizes of liposomes. (D) Mean fluorescence intensity of inflamed paws (n = 3; mean ± SD) of CIA model mice at different time points after liposomes were injected intravenously. (E) Targeting index (TI) of inflamed joints in CIA model mice at 48 h after injection of liposomes with different sizes. (F) Fluorescence images of biodistribution in CIA model mice 48 h after intravenous injection with liposomes of different sizes.

Effects of liposome surface charge on pharmacokinetics, in vivo targeting efficiency, and biodistribution DiD-loaded liposomes with different surface charge (negative, slightly negative, slightly positive, and positive) were injected into healthy ICR mice to investigate their pharmacokinetics. The results showed that liposomes with a slightly negative surface charge had higher AUC0→t than other liposome types, while T1/2 and MRT0→t were not significantly different among all groups. (Figure 4A-B). The relatively shorter T1/2 of negative, and lower AUC0→t of positive liposomes may be due to the fact that the liposomes with high charge density are more easily recognized by complement proteins and consequently cleared by the RES system47-49. In addition, compared to liposomes with slight negative charge, liposomes with slight positive charge (with lower AUC0→t and shorter T1/2) are more likely to interact with serum proteins and blood cells, which may attenuate their circulation time in the blood50-51. To evaluate the effect of surface charge on liposomes’ inflammatory joint targeting capacity, DiD-labeled liposomes with varying surface charge were administrated intravenously to CIA model mice (n = 3). Fluorescence intensities of inflamed paws were determined by in vivo fluorescence imaging. Live images of hind paws showed that the fluorescence signals of liposomes with slight negative charge was visibly higher than that of the other three groups (Figure 4C). Quantitative analysis (Figure 4D) indicated that liposomes with slight negative charge produced the highest mean fluorescence intensity throughout the test period, reaching their maximum at 12 h. At 48 h post-injection, the targeting indexes indicated that liposomes with slight negative charge have stronger RA targeting (Figure 4E). The superior RA targeting of liposomes with ACS Paragon Plus Environment

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slight negative charge might be due to their interaction with synoviocytes37, although the pharmacokinetic parameters among all groups were not significantly different. The biodistributions of liposomes bearing various surface charges were estimated by fluorescence images of the organs and inflamed paws, obtained 48 h post-injection. The results indicated that the inflamed paws of mice given liposomes with slight negative charge had the strongest fluorescent signals among all experimental groups. These images also revealed that positive and slightly positive liposomes are prone to accumulate in the liver due to their affinity for hepatocytes52 (Figure 4F). It can be concluded from the results that positive liposomes were easily adsorbed by complement proteins, engulfed by RES, and accumulated in the liver tissue, while liposomes with strong negative charge were easily able to activate the complement system. Liposomes with slightly negative charge had the best in vivo RA targeting effect.



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Figure 4. Pharmacokinetic, in vivo targeting, and biodistribution of liposomes carrying different surface charges. (A-B) Pharmacokinetic profiles of liposomes with varying surface charge in healthy mice (mean ± SD, n = 3). AUC, area under the fluorescence intensity-time curve. T1/2, half-life. MRT, mean residence time. (C) In vivo fluorescence imaging of inflamed paws (AI score>3) (left, A) and normal paws (right, NA) in CIA model mice after intravenous injection of liposomes carrying different surface charges. (D) Mean fluorescence ACS Paragon Plus Environment

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intensity of inflamed paws (n = 3; mean ± SD) of CIA model mice at different time points after liposomes were injected intravenously. (E) Targeting index (TI) of inflamed joints in CIA model mice at 48 h after injection of liposomes with different surface charges. (F) Fluorescence images of biodistribution in CIA model mice, 48 h post-injection with liposomes carrying different surface charges. Effects of PEG chain length in liposomes on pharmacokinetics, in vivo targeting efficiency, and biodistribution DiD-liposomes prepared with PEG of different chain lengths (1 kDa, 2 kDa, 5 kDa, and 10 kDa) were injected into healthy ICR mice to investigate the effects of PEG chain length on the pharmacokinetics of liposomes. The study demonstrated that liposomes modified with 5 kDa PEG had relatively better long-term circulation parameters, including higher AUC0 → t, and longer T1/2 and MRT0 → t in all experimental groups. Long-term circulation effects were enhanced as PEG chain lengths increased from 1 kDa to 5 kDa. However, upon reaching 10 kDa, the trend began to reverse (Figure 5A-B), possibly due to formation of curved micelles caused by excessively long PEG chains. This kind of micelle is more likely to cause aggregation or destruction of liposomes, thus reducing the blood circulation time53. In addition, liposomes decorated with excessively long PEG chains may inhibit uptake of liposomes by targeted cells, and recognition by protein receptors54. Moreover, longer PEG chains will influence release of therapeutics from the nanocarrier55. Therefore, 1 kDa, 2 kDa, and 5 kDa chain length PEG stocks were used to prepare DiD liposomes to test in vivo targeting. To investigate the effects of PEG chain length in liposomes on inflamed joint targeting, DiD-labeled liposomes with various PEG chain lengths were administrated intravenously into CIA model mice (n = 3). The fluorescence signal of paws was measured by in vivo fluorescence imaging. Live images of hind paws showed that the fluorescence signal of 5 kDa PEG decorated liposomes was visibly stronger than that of the other groups (Figure 5C). This can also be seen in the quantitative analysis (Figure 5D). Liposomes with 5 kDa PEG produced the highest intensity fluorescence throughout the test period, reaching its maximum at 24 h, which can be attributed to the longer circulation time of liposomes with 5 kDa PEG. The targeting index reflected ACS Paragon Plus Environment


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the same trend: 5 kD PEG liposomes have excellent RA targeting (Figure 5E). Specifically, liposomes with a PEG chain length of 5 kDa have good “anti-opsonization” effects and don’t induce formation of curved micelles, as can happen with excessively long PEG hydrophilic head groups53. Liposomes with 5 kD PEG yield optimal long-circulation times and inflamed joint targeting. Biodistribution of liposomes with different PEG chain lengths was estimated using fluorescence images of the organs and inflamed paws, obtained at 48 h post-injection. The results showed that the inflamed paws of mice treated with 5 kDa PEG liposomes had stronger fluorescent signals than those of the other groups (Figure 5F). These images also confirm that longer circulation times of liposomes correlate with greater accumulation in inflamed joints. In general, we could see that liposomes modified with 5 kD PEG have longer circulation times and less impact on surface properties56. Thus, 5 kDa was the optimal PEG chain length for liposomes in RA targeting therapy.


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Figure 5. Pharmacokinetics, in vivo targeting, and biodistribution of liposomes with different PEG chain lengths. (A-B) Pharmacokinetic profiles in healthy mice of liposomes with various PEG chain lengths (mean ± SD, n = 3). AUC, area under the fluorescence intensity-time curve. T1/2, half-life. MRT, mean residence time. (C) In vivo fluorescence imaging of inflamed paws (AI score>3) (left and right, A) in CIA model mice after intravenous injection of different PEG chain length liposomes. (D) Mean fluorescence intensity of inflamed paws (n = 3; mean ± SD) of CIA model mice at different time points after liposomes were injected ACS Paragon Plus Environment


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intravenously. (E) Targeting index (TI) of the inflamed joint in CIA model mice at 48 h after injection of liposomes with different PEG chain lengths. (F) Fluorescence images of biodistribution in CIA model mice 48 h after injection of liposomes with different PEG chain lengths. Effects of liposome PEG content on pharmacokinetics, in vivo targeting efficiency, and biodistribution DiD-liposomes modified with varying concentrations (5%, 10% and 20% w/w of total lipid) of 5 kDa PEG were injected into healthy ICR mice to assess the effects of PEG (5 kDa) content on liposome pharmacokinetics. The results indicate that liposomes with 10% and 20% PEG content (5 kDa) have similar pharmacokinetic profiles, with longer circulation times than liposomes with 5% PEG (Figure 6A). Five percent PEG (5 kDa) also had the worst in vivo circulation properties, and there was no significant difference in AUC0→t between liposomes with10% or 20% PEG (5 kDa). However, the T1/2 and MRT0→t values were better for liposomes containing 10% PEG than those containing 20% (Figure 6B). In addition, use of excessive PEG might promote formation of curved micelles or unstable liposome structures53. Accordingly, liposomes modified with 10% PEG (5 kDa) had long circulation times in vivo which were not inferior to those of the 20% group. To evaluate the influence of PEG content on inflammatory joint targeting, DiD-labeled liposomes made with various PEG concentrations were administrated intravenously into CIA model mice (n = 3). Fluorescence intensities of paws were determined by in vivo fluorescence imaging. Live imaging of hind paws revealed that 10% and 20% PEG liposomes produced fluorescence intensities that were similar to each other, and stronger than that of the 5% PEG group, especially at the last three time points (Figure 6C). Quantitative analysis of mean fluorescence intensity (MFI) revealed that the 5% PEG liposome group had strong fluorescence intensity in the paws until the 6-h point. After that, the 10% and 20% PEG liposomes demonstrated better RA-targeting (Figure 6D). Targeting Index evaluations showed that liposomes with 5%, 10% and 20% PEG content (5 kD) had similar RA targeting effects, which indicated PEG content (within a limited range) might not be crucial to RA targeting (Figure 6E). ACS Paragon Plus Environment

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The biodistribution of liposomes with varying PEG content was estimated using fluorescence images of organs and inflamed paws 48 h after initial injections. The results showed that inflamed paws of mice treated with 10% and 20% PEG liposomes had stronger relative fluorescence than those of the 5% PEG group (Figure 6F). According to these results, liposomes containing 10% or 20% PEG had similar fates in vivo, and similar RA targeting distribution. Considering that increasing PEG content may hinder drug release56 and interactions of liposomes with target cells and ligands57, 10% was chosen as optimal PEG content.



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Figure 6. Pharmacokinetic, in vivo targeting, and biodistribution of liposomes with varying PEG content. (AB) Pharmacokinetic profiles of liposomes with varying PEG content in healthy mice (mean ± SD, n = 3). AUC, the area under the fluorescence intensity-time curve. T1/2, half-life. MRT, mean residence time. (C) In vivo fluorescence imaging of normal paws (left, NA) and inflamed paws (AI score>3) (right, A) in CIA model mice after intravenous injection of liposomes with varying PEG content. (D) Mean fluorescence intensity of inflamed paws (n = 3; mean ± SD) of CIA model mice at different time points after liposomes were ACS Paragon Plus Environment

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intravenously injected. (E) Inflamed joint targeting index (TI) in CIA model mice 48 h after injection of liposomes with varying PEG content. (F) Fluorescence images of biodistribution in CIA model mice 48 h after injection with liposomes carrying different PEG levels. In vitro release profile of Dex liposomes Kinetics of in vitro release of Dexamethasone from Dex liposomes in PBS (pH 7.4) indicated that 47.38% of Dex was released from liposomes in 4 h, meanwhile 85.51% of Free Dex was released (Fig. 8). These results suggest that Dex liposomes have better in vivo sustained release behavior, which might be beneficial in RA targeting treatment. 100

Cumulative release of dexamethasone (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

Dex Liposomes

40

Free Dex

20 0 0

6

12

18

24

30

36

42

48

Time (h)

Figure 7. In vitro Dex release curve of Dex liposomes and Free Dex in PBS (mean ±SD, n = 3) In vivo therapeutic efficacy of Dex liposomes The CIA model has been established as the most extensively used RA animal model to study pathogenic mechanisms of RA, and potential therapeutic efficacy of drugs delivered by liposomes58. Dexamethasoneloaded liposomes (Dex Liposomes) were prepared by an optimized formulation and process. CIA model mice (6 mice per group) were injected with Dex liposomes, Free Dex, or PBS, once every 2 days for 20 days beginning after the arthritis index of each mouse was greater than or equal to 4. Changes in the arthritis index were demonstrated in Figure 8A. Compared with the PBS group, the Free Dex group could effectively inhibit the pathological progress in inflamed paws. Treatment with Dex Liposomes could significantly ACS Paragon Plus Environment


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further decrease the arthritis index. Figure 8B provides images of normal and inflamed paws after 20 days of treatment. Compared to normal paws, the paws of the CIA model mice treated with PBS showed significant swelling. In the Free Dex group, swelling was greatly reduced, and only a little redness could be observed on the paws. Compared with the normal group, the paws in the Dex Liposome group exhibited the least morphological change, appearing almost recovered, which proved that Dex Liposomes had the strongest anti-inflammatory effect and/or targeting for treatment of RA. Microcomputed tomography analysis Cartilage erosion and bone destruction are the critical pathological symptoms of RA by which severity of the disease is evaluated59. Bone damage in all groups was evaluated by Micro-CT. As shown in Figure 8C, severe bone and cartilage erosion occur around the inflamed toe and ankle joints in the PBS group, while only minor bone destruction was observed in toe joints in the Free Dex group. In the Dex Liposomes group, no significant pathological changes of bone and cartilage were seen, indicating that Dex Liposomes enhanced RA-targeting, increasing accumulation of Dex in the inflammatory toe and ankle joints, and thus greatly improving treatment efficacy.

Figure 8. Therapeutic efficacy of Dex-loaded liposomes in CIA model mice. (A) Averaged arthritis index of CIA model mice after first dose post-CIA model establishment. (B) Optical photo of inflamed paws from CIA model mice after treated with PBS, Free Dex or Dex Liposomes and normal DBA/1 mice without any therapy. (C) Micro-computed tomography (micro-CT) results of normal paws and inflamed paws after treated with ACS Paragon Plus Environment

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PBS, Free Dex, or Dex Liposomes. Histological analyses The effect of Dex Liposome treatment on lesions and bone and cartilage damage levels was evaluated by histological analysis of H&E and immunohistochemical stained tissues. At the conclusion of treatment, ankle joints were subjected to slitting to evaluate damage to bone and cartilage, and to determine expression levels of some inflammatory factors. As the first line of Figure 9 shown, the joint cavity sections of the PBS group showed obvious synovial hyperplasia with bone and cartilage damage. These symptoms were alleviated in the Free Dex group. In the Dex liposomes group, neither bone and cartilage erosions, nor synovial hyperplasia were found. Immunohistochemical results demonstrated that relative to the normal group, expression of TNFα, IL-6, IL-1β, and CD31 were obviously upregulated in the PBS and Free Dex groups. However, the levels of these cytokines were apparently reduced of Dex Liposomes group (Figure 9). These images indicate that liposomes may significantly increase the therapeutic effect of Dex by ameliorating the inflammatory response and vascular proliferation in the inflamed joints of RA.



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Figure 9. Histological images of ankle joints of normal and CIA model DBA/1 mice treated with PBS, Free Dex or Dex Liposomes. Various degrees of neovascular tissue (arrows) and bone erosion (*) are seen in PBS, Free Dex, and Dex liposomes groups (H&E staining, 100×). Immunohistochemistry images show various levels of TNF-α, IL-1β, IL-6, and CD31 in all groups (100×). (B represents bone, C represents cartilage, and S represents synovial tissue) FACS analysis of fluorescent liposome internalization by synoviocytes in CIA model mice Endothelial vascular proliferation in inflamed synovial membranes in RA produces highly permeable vascular tissue (a pannus) similar to a solid tumor60 at the synovial site, which allows nanocarriers to enter the joint cavity. In the present study, DiA liposomes with uniform fluorescence intensity were intravenously injected ACS Paragon Plus Environment

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into normal and CIA DBA/1 mice, respectively. After 12 h, fluorescence intensity of whole mice and hind paws were obtained from in vivo images using a NIRF imaging system. Real-time fluorescence images showed that fluorescence intensity in the paws of CIA model mice was obviously stronger than in the paws of normal mice. Quantitative analysis indicated that NIRF intensity in CIA model mice was 2.1-fold higher than in normal mice (Figure 10A-B). These results revealed the high permeability of synovial endothelial vessels in CIA model mice to the optimized liposomes. Another goal of this experiment is to identify the inflammatory cells that play an important role in retention of the liposomes in inflamed joints. As shown in Figure 11A-C, FACS analysis showed that uptake of DiA liposomes by macrophages, fibroblasts, and endothelial cells in the CIA group was elevated compared with the normal group. This elevation was especially pronounced in fibroblasts and macrophages (Figure 11D). This analysis also indicated that the retention effect in RA inflamed paws was mainly due to uptake by various inflammatory cells. This information can be used as a critical foundation for design of active inflammationtargeting drug delivery systems. These results demonstrate that induction of CIA in mice by causing vascular inflammation could increase vascular permeability in the inflamed joints, namely the ELVIS effect. Thus the high retention of nanocarriers such as liposomes, could be mainly due to their uptake by activated macrophages61 and fibroblasts.

Figure 10. (A) In vivo fluorescence imaging of normal and CIA model DBA/1 mice (n = 3) at 12 h after ACS Paragon Plus Environment


injection. (B) Fluorescence intensity of normal and inflamed paws (n = 3, mean ± SD).


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Figure 11. Comparison of fluorescence-activated cell sorting analysis of synovial cells released from hind limb joints in Normal DBA/1 mice and CIA model mice 12 h after intravenous injection. (A) F4/80+ cells (macrophages). (B) CD31+ cells (endothelial cells). (C) vimentin+ cells (fibroblasts). (D) Mean NIRF intensity of different synovial cells in normal and inflamed paws (n = 3; mean ± SD) *represents significant difference between F4/80+ cells from normal and CIA model DBA/1 mice, (p< 0.05), ***represents significant difference between Vimentin+ cells from normal and CIA DBA/1 mice, (p< 0.001). Discussion Liposomes have been extensively used to delivery therapeutic drugs for RA treatment due to their easy preparation, biocompatibility, versatility to be modified with various ligand and loading capacity to hydrophobic and hydrophilic drugs. The physical and chemical characteristics of liposomes have significant influence on their fate in vivo. They can affect not only in vivo circulation time and transport through multiple biological barriers, but also retention ability of the site targeted by the liposomes62. In the present research, we studied the effects of liposome particle size, surface charge, and PEG modification on their circulation time and in vivo targeting ability for RA treatment. The results demonstrated that liposomes with 100 nm particle size, slightly negative charge, and 10% PEG5000 had optimal in vivo half-life and inflamed joint targeting capacity. Systematic analysis of pharmacokinetic results indicates that the most important property for long in vivo half-life during passive targeting of RA may be particle size and PEG chain length, while surface charge and PEG chain length had a stronger influence on RA site retention. Dexamethasone loaded liposomes with an optimized formulation could significantly improve therapeutic efficacy. This study provides a strong foundation for future studies of liposome-based treatments targeting RA. In addition, we explored the mechanism of liposome retention in inflamed joints in RA, which was different from their retention mechanism in solid tumors. The results elucidated that the phagocytic abilities of synovial fibroblasts and macrophages were significantly increased after activation. This possible new retention mechanism provides a theoretical basis for future design of a RA targeting drug delivery system. ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Materials. Egg yolk lecithin and cholesterol were provided by A.V.T pharmaceutical Co., LTD. (Shanghai, China). 1,2-dioleoyl3-trimethylammonium-propane (DOTAP) was purchased from Corden Pharma Switzerland LLC (Liestal Switzerland). DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG5000, and DSPE-PEG10000 were purchased from Ponsure Biotechnology (Shanghai, China). Dexamethasone and Deoxyribonuclease type I were purchased from Dalian Meilun Biotechnology Co., Ltd (Dalian, China). Type IV Collagenase was purchased from Sigma-Aldrich (St. Louis, MO, USA). Type II bovine collagen, and complete and incomplete Freund's adjuvant were purchased from Chondrex, Inc. (Redmond, WA, USA). 1,1’dioctadecyl-3,3,3’,3’–tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) and 4-(4(Dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) were purchased from Fanbo biochemical Co., Ltd. (Beijing, China). Other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Animals. 25-30g male ICR mice and 8-weeks old DBA/1 male mice were obtained from Sino-British SIPPR/BK Lab Animal Co., Ltd. (Shanghai, China). All animal experiments were consistent with the Guiding Principles for the Care and Use of Experimental Animals in Fudan University (Shanghai, China). The experimental protocols of this article were estimated and approved by the Ethics Committee of Fudan University. Preparation of liposomes. For pharmacokinetic studies, liposomes of different sizes, charges, PEG chain lengths, and PEG concentrations were prepared by a film dispersion and extrusion method. Briefly, 25 mg Egg yolk lecithin (EPC), 2.5 mg Cholesterol, 2.5 mg PEG2000, and 25 µg DiD (5 mg/mL in 100% acetone) were dissolved in 6 mL dichloromethane and evaporated in rotary evaporator, yielding a thin, homogeneous lipid film. Dried films were hydrated in purified water with magnetic stirring for 15 minutes, then extruded through polycarbonate membranes with a range of pore sizes to obtain liposomes with different particle sizes. Membranes with 400 nm, 200 nm, 100 nm, and 50 nm pore sizes were used to generate liposomes with average ACS Paragon Plus Environment


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diameters of 350 nm, 200 nm, 100 nm, and 70 nm, respectively. Different amounts (0, 1, 1.5, 2 mg) of DOTAP were added to the formulation above to form a uniform lipid film. After hydration with purified water for 15 min, lipid suspensions were extruded through 100 nm pore size polycarbonate membranes to obtain liposomes with various surface charges (positive charge, negative charge, slight positive charge, and slight negative charge). Various PEG chain lengths (1 kDa, 2 kDa, 5 kDa, and 10 kDa), and different PEG amounts (1.25, 2.5, and 5 mg) were used to prepare liposomes with different surface hydrophilic properties. After liposome preparation, unbound DiD was removed on a Sephadex column. For in vivo imaging and biodistribution studies, different particle size, surface charge, and PEGylated liposomes were prepared via the method described above except that amounts of DiD (5 mg/mL in 100% acetone) in the formulation was increased from 25 µg to 125 µg. For the pharmacodynamic study, Dex was loaded into the liposomes using a film hydration and ultrasonication method designed to control particle size. Free Dex was removed using a Sephadex column. Optimized amounts of DOTAP, PEG chain length, and PEG content were used in the formulation. Other lipids and processes were consistent with those described above except that 25 µg DiD (5 mg/mL in 100% acetone) was replaced by 1 mg Dex.

Liposomes were prepared for fluorescence-activated cell sorting (FACS) analysis by a film hydration and ultrasonication method designed to control particle size. Formulation and process conditions were consistent with those used in the pharmacodynamic studies, except that 1 mg Dex was replaced with 125 µg DiA (5 mg/mL in 100% acetone). Characterization of liposomes. The mean particle size, polydispersity index (PDI), and zeta-potential of liposomes were measured by a dynamic light scattering (DLS) detector (Nano-ZS, Malvern, UK). Transmission electron microscopy (TEM, Tecnai G2 F20 S-Twin, FEI, USA) was used to evaluate the morphology of the liposomes For TEM, liposomes were dripped onto copper grids and stained with uranyl ACS Paragon Plus Environment

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acetate (2%, w/v). Encapsulation efficiency (EE) and drug loading (DL) of liposomes were determined using the formulas below: EE (%) = DL (%) =

Total drug ― Unencapsulated drug Total drug Total drug ― Unencapsulated drug Weight of liposomes

× 100%

(1)

× 100%

(2)

The unencapsulated drug was obtained by ultrafiltration, and quantitated by HPLC after dissolving in methanol. Total drug load in liposomes was determined by HPLC, after de-emulsification with methanol. HPLC assays for dexamethasone were performed on a DIKMA C18 HPLC column (4.6 mm × 250 mm, 5 µm) at 30℃ with a sample injection volume of 20 µL. The mobile phase consisted of water-acetonitrile (50:50, v/v). The flow rate was 1 mL/min, and the detection wavelength was 240 nm. Induction of the RA model. CIA was induced in DBA/1 mice as previously described63. Briefly, type II bovine collagen was dissolved at 10 mg/mL in 0.1 N acetic acid with vortexing. Then, an equal volume of complete Freund's adjuvant (CFA) was added and fully emulsified with the collagen solution in an ice bath. Male DBA/1 mice (8 weeks) were injected subcutaneously at the tail root with 100 μL of the 1:1 collagen:adjuvant emulsion. After three weeks, mice were given a booster injection with 100 μL of the same emulsion. The progression of arthritis was then monitored daily, and onset of RA was determined based on paw inflammation and swelling. The severity of arthritis was scored by comparing each mouse’s arthritis index(AI) with a published standard64, in which 0 = normal appearance, 1 = inflammation and edema in one toe, 2 = inflammation and edema in two or more toes, but not the entire paw, 3 = inflammation and edema extend to the entire paw, and 4 = inflammation and edema are present throughout the paw and ankle. Four paws were scored separately, resulting in a summative clinical score of 16 for each animal. Customarily, an arthritis index of 4 or more indicates that CIA has been successfully modeled. Studies of liposome pharmacokinetics. Pharmacokinetic studies were carried out on male ICR mice (25–30 g) using DiD dye to label liposomes as previously described65. To determine liposome half-lives, 200 μL/mouse of various formulations of DiD-liposomes were intravenously injected. Fifty microliter ACS Paragon Plus Environment


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samples of blood were collected at time points of 2, 5, 15, and 30 min, and 1, 3, 6, 9, 24, and 48 h after injection. Obtained blood samples were diluted 5 times in PBS, and their fluorescence intensities were determined in a microplate reader. (640 nm/680 nm). In vivo imaging and biodistribution of liposomes. The accumulation of various formulations of liposomes at sites of joint inflammation was visualized using near-infrared fluorescence (NIRF) imaging. Briefly, 0.2 mL of DiD-labeled liposomes with different physical and chemical properties were intravenously injected into normal or CIA model DBA/1 mice to investigate their biodistribution by in vivo imaging system (PerkinElmer, USA) at 1, 3, 6, 12, 24, and 48 h. Subsequently, mice were sacrificed and dissected to obtain the main organs and the hind limbs. The biodistribution of liposomes was visualized and quantified by determining fluorescence intensities at regions of interest (ROI) using the in vivo imaging system described above. The targeting index (TI) was determined for each experimental group by taking the ratio of fluorescence intensity at the inflamed joint to total fluorescence intensity of the liposomes delivered by intravenous injection according to the formula below: fluorescence signal of inflamed joint

TI (%) = fluorescence signal of the injected dose of liposomes × 100%

(3)

Study of in vitro release of dexamethasone from liposomes. Dexamethasone release from liposomes in dialysis was investigated by methods described previously66-67. One milliliter of Dex liposomes (1 mg/mL) or Free Dex (dissolved in 30% (v/v) PEG 400, 1 mg/mL) was added into a dialysis bag with a molecular weight cutoff of 3,500 KD. Dialysis bags were then placed in glass bottles containing 100 mL of release medium (PBS, pH 7.4), incubated at 37°C and 50 rpm. One mL of medium was taken at 10, 20, and 30 min, and 1, 2, 3, 4, 6, 12, 24, and 48 h, and an equal-volume of dissolution medium was added to the bottle after sampling. Samples were then assayed for dexamethasone by HPLC. Drug release from liposomes was plotted against time. Pharmacodynamics study of liposomes. CIA model mice (arthritis index ≥ 4) were randomly divided into three groups (n = 6) and were intravenously injected with PBS, free dexamethasone (Free Dex) (dissolved ACS Paragon Plus Environment

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in 10% ethanol, v/v) or dexamethasone-liposomes (Dex Liposomes) at a dexamethasone dosage of 1 mg/kg6769

once every two days for 20 days. Normal DBA/1 mice were used as a control group. The Arthritis Index

(AI) of each paw in every group was recorded every other day and on day 20 after arthritis induction (the study endpoint). Micro-computed tomography (Micro-CT) study of liposomes. The effects of therapy with Dex liposomes were examined post-treatment using micro-CT as previously described70. The hind limb from the experimental and normal group were isolated and scanned with an in vivo Micro-CT imaging system (Skyscan 1176) (Bruker, Germany) after fixing with 4% paraformaldehyde for at least 24 h. The experimental parameters of this study were as follows: source voltage: 45 kV, source current: 556 μA, image pixel size: 8.7 μm, filter: aluminum 0.2mm. CT-Vol software (Bruker, Germany) was used to reconstruct the Micro-CT images. Histological analysis. After 20 days of treatment, mice hind limb of all groups were dissected for histological analysis. After fixed with 4% paraformaldehyde for at least 24 h, the paws were decalcified with 15% EDTA PBS solution. After that, samples were dehydrated using a range of concentrations of the ethanol solution. Next, the paw samples were paraffin-embedded and sliced into 4 μm thick sections using a microtome for hematoxylin-eosin (H&E) staining. For immunohistochemical studies, tissue sections of all groups were incubated with specific primary antibodies of TNF-α, IL-1β, IL-6, and CD31, followed by suitable secondary antibodies after antigen retrieval and serum blocking. Fluorescence-activated cell sorting analysis of cells isolated from joint tissue. DiA-loaded liposomes were delivered to CIA and normal DBA/1 mice by tail vein injection. After 48 h, inflamed paws were surgically removed with a scalpel and minced aseptically. Tissues were further digested with PBS containing collagenase type IV (1 mg/mL) and DNAase Type I (0.15 mg/mL) at 37°C for 20 minutes. A single-cell suspension was obtained by passage through a 75 μm screen cloth. For FACS evaluation, cells were incubated in the dark with primary antibodies to CD45 (PerCP-Cy5.5, ACS Paragon Plus Environment


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ebioscience), F4/80 (FITC, BD), Vimentin (Alexa Fluor®647, Cell Signaling) and CD31 (eFluor®450, ebioscience) for 30 minutes, then washed and analyzed by flow cytometry. Statistical analysis. Experimental results were analyzed with GraphPad Prism 6.0 (GraphPad Software, Inc. La Jolla, CA, USA). Statistical differences were evaluated using unpaired Student's t-test for comparison of two groups, and one-way ANOVA for multiple-group comparisons. The data were expressed as mean ±SD, and a p-value

Role of Liposome Size, Surface Charge, and PEGylation on

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