In Situ Size Amplification Strategy Suppresses Lymphatic Clearance for Enhanced Arthritis Therapy

In Situ Size Amplification Strategy Suppresses Lymphatic Clearance for Enhanced Arthritis Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Situ Size Amplification Strategy Suppresses Lymphatic Clearance for Enhanced Arthritis Therapy Xianyan Qin, Luhan Zhang, Yang-Bao Miao, Linxi Jiang, Liang Zou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5069556/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 12 You are reading this latest preprint version Abstract Rheumatoid arthritis (RA) is an autoimmune condition causing painful swelling and inflammation due to immune system attacks on healthy cells. However, arthritic sites often experience increased lymph flow, hastening drug clearance and potentially reducing treatment effectiveness. To address this challenge, an in situ size amplification has been proposed to inhibit lymphatic clearance and thereby enhance arthritis therapy. This system has been developed based on a conjugate of dexamethasone (Dex) and polysialic acid (PSA), linked via an acid-sensitive linker, supplemented with bis-5-hydroxytryptamine (Bis-5HT) on the PSA backbone. Under physiological conditions, the system autonomously assembles into stable nanoparticles (PD5NPs), facilitating prolonged circulation and targeted delivery to inflamed joints. Upon arrival at arthritic joints, Bis-5HT reacts to elevated myeloperoxidase (MPO) levels and oxidative stress, prompting particle aggregation and in-situ size amplification. This in situ size amplification nanocarrier effectively inhibits lymphatic clearance and serves as reservoirs for sustained Dex release in acidic pH environments within arthritic sites, thus continuously alleviating RA symptoms. Moreover, investigation on the underlying mechanism elucidates how the in situ size amplification nanocarrier influences the transportation of PD5NPs from inflamed joints to lymphatic vessels. Our study offers valuable insights for optimizing nanomedicine performance in vivo and augmenting therapeutic efficacy. Rheumatoid arthritis nanomedicine delivery in situ size amplification aggregates lymphatic clearance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction As a chronic and uncurable autoimmune disease, rheumatoid arthritis (RA) often requires long-term or even lifelong medication [ 1 , 2 ]. Nanomedicines have been widely recognized to enhance delivery efficiency to inflamed sites and improve therapeutic index, thereby reducing administration frequency [ 3 , 4 ]. As we known, when administrated in vivo, drug formulations will go through absorption, distribution, metabolism and excretion processes, which play a crucial role on their in vivo safety and effectiveness [ 5 ]. Currently, most of the attention have been focused on enhancing targeted distribution in inflamed sites when developing nanomedicine for treating chronic inflammatory diseases [ 6 ], very less on slowing down the clearance rate of nanomedicines. In fact, the increased lymph flow and excessive lymph angiogenesis in arthritic sites driven by overactivated immune response will lead to accelerated clearance of nanomedicines from lymphatic system [ 7 , 8 ]. To prolong therapeutic efficacy and reduce frequency of drug administration in RA treatment, it is necessary to simultaneously achieve targeted delivery and delayed drug clearance in inflamed sites. The joint-space residence time of nanoparticles is heavily influenced by their clearance rate from lymphatic system in synovium [ 9 ]. The particle size plays a pivotal role on the transport and clearance process from inflamed synovium to lymphatic system [ 10 ]. Upon systemic administration, nanoparticles enter the joint space via the highly vascularized capillary network of the inflamed sub-synovium and they are primarily cleared from the joint space via the lymphatic system [ 11 ]. Generally, nanoparticles smaller than 10 nm are swiftly transported to the blood capillaries, while those within the range of 10–100 nm effectively distribute in lymphatic capillaries [ 12 , 13 ]. Larger nanoparticles might encounter bigger steric hindrance within the gel-like extracellular matrix of joint tissues and exhibit much slower diffusion rate towards lymphatic vessels [ 14 ]. Conversely, nanoparticles with larger sizes often exhibit poor in vivo circulation and rapid phagocytosis by the reticuloendothelial system (RES) [ 15 , 16 ]. To address this dilemma, an in situ size amplification drug carrier that maintains a small particle size in normal physiological condition while undergoes site-specific size amplification upon reaching inflamed sites, can undoubtedly fulfill the diverse therapeutic demand. Recently, the Xu group designed and synthesized a self-expanding nanogel with multicompartment structure, which was stable in physiological environment while became larger under acidic pH and redox condition, thereby attenuating the side effects and boosting synergistic anticancer effect [ 17 ]. Although these nanocarrier system were effective in cancer therapy, it might not be directly utilized to inflammatory conditions. It is worth noting that nanocarriers used in chronic inflammatory diseases should have good biocompatibility and not cause any immune activation or inflammatory responses [ 18 ]. In our previous work, we designed a transformable nanoparticle which underwent shape transformation from nanoparticles to nanofibers under acidic pH and ligand-receptor interaction, enabling prolonged retention in inflammatory joints [ 19 ]. However, the re-assembly of KLVFF peptide in this transformable nanoparticle was very susceptible to external mechanical stimuli such as the shear stress in blood flow, posing unpredictable risk during in vivo circulation. Therefore, nanocarriers can undergo size amplification in response to inflammatory stimuli with high specificity will be an efficient and safe strategy. In the RA microenvironment, an abundance of neutrophils is recruited to drive the RA progression by secreting a large number of inflammatory cytokines, chemokines and enzymes [ 20 , 21 ], among which myeloperoxidase (MPO) is an azurophilic granule enzyme abundantly expressed by neutrophils [ 22 ]. MPO can catalyze the conversion of phenolic residues into free radical, contributing to inflammatory response and tissue damage in RA [ 23 , 24 ]. According to the high level of MPO in inflamed joints and its catalytic characteristic, we proposed an MPO-responsive in-situ size amplification strategy aimed at impeding drug clearance from lymphatic system to ultimately extend therapeutic efficacy. Herein, we synthesized a conjugate consisted of anti-inflammatory dexamethasone (Dex) and naturally occurred polysialic acid (PSA) via an acid-sensitive linker, and modified bis-5-hydroxytryptamine (Bis-5HT) moieties on the PSA backbone (Fig. 1 A). The formed conjugates, termed PSA-Dex-Bis-5HT, can self-assemble into stable nanoparticles (PD5NPs) in physiological condition and facilitate in vivo circulation and targeted delivery to inflamed sites. When reaching arthritic joints, Bis-5-HT can respond to the local MPO/hydrogen peroxide and induce the aggregation of PD5NPs or binding to neighboring extracellular matrix protein via radial formation, resulting in in-situ size amplification and impaired transportation of drug formulation via lymphatic vessels. Furthermore, these aggregates can serve as drug depots for sustained Dex release under local acidic pH in arthritic joints, persistently mitigating RA (Fig. 1 B). In our study, the MPO responsive in situ size amplification behavior of PD5NPs was confirmed and the in vivo performance of PD5NPs, encompassing pharmacokinetics, biodistribution, transportation and therapeutic efficacy were systematically investigated. More importantly, we extensively elucidated how the size amplification behavior affected the transportation of PD5NPs from inflamed joints to lymphatic vessels, and revealed the effectiveness of restraining lymphatic clearance for prolonged RA remission. To our knowledge, this in-situ size amplification strategy has never been applied in the treatment of inflammatory diseases. Our findings can not only offer an effective strategy for RA treatment, but also provide a new strategy to optimize the in vivo performance of nanomedicine. 2. Materials and methods 2.1. Materials Fmoc-Glu-OH was purchased from Bide Pharmatech Ltd. (Shanghai, China). 5-hydroxytryptamine (5-HT) was purchased from Sigma-Aldrich (Chengdu, China). 1-ethyl-3-(3-dimethyl amino propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Macklin (Shanghai, China). Myeloperoxidase (MPO) was purchased from Sino Biological Inc. (Beijing, China). Dexamethasone (Dex) and hydrogen peroxide (H 2 O 2 ) were obtained from Aladdin Reagent, Ltd (Shanghai, China). Polysialic acid (PSA) was bought from Carbosynth China Ltd (Suzhou, China). Cy5.5-NH 2 was obtained from Meilun (Dalian, China). Complete Freund's adjuvant (CFA) was provided by Chondrex (Washington DC, USA). All reagents and solvents were purchased from Kemiou (Tianjin, China). Male SD rats were provided by Dashuo experimental animal center (Chengdu, China). All animal studies were approved by the Ethics committee of Sichuan Provincial People's Hospital. 2.2. Synthesis of Bis-5HT-Glu-Fmoc: Briefly, N-hydroxysuccinimide (3 mmol) and EDC (3 mmol) were mixed with Fmoc-Glu-OH (1 mmol) in dimethylformamide (DMF, 30 mL) at room temperature and stirred for 30 min. Subsequently, 5-hydroxy tryptophan (5-HT, 2 mmol) and triethylamine dissolved in DMF (5 mL) were introduced into the above mixture. The reaction mixture was stirred for 2 h. The product was purified via reversed-phase chromatography and validated by 1 HNMR (400 MHz, Bruker AMX-400, USA). 2.3. Synthesis of Bis-5HT-Glu-NH 2 Bis-5HT-Glu-Fmoc (1 mmol) dissolved in 30 mL of DMF was mixed with piperidine (5 mmol) under stirring, and the mixture was stirred at room temperature for 2 h. DMF was removed under reduced pressure, and the residue was subsequently purified by reversed-phase chromatography to yield the compound Bis-5HT-Glu-NH 2 . The final product was obtained and confirmed by 1 HNMR analysis. 2.4. Synthesis of PSA-Dex-Bis-5HT Initially, Dex-NH 2 was synthesized according to previous established procedure [ 25 ]. Dex (5 mmol) and hydrazine hydrate (15 mmol) were dissolved in 30 mL of methanol, and a slow addition of 30 µL acetic acid preceded a reflux reaction at 80°C for 5 h. After the removal of unreacted substances through dialysis against ethanol and deionized water, Dex-NH 2 was obtained through lyophilization and subsequently dissolved in DMSO for subsequent reaction. In a parallel reaction, PSA (0.3 mmol disaccharide repeats), EDCI (1.5 mmol), and NHS (1.5 mmol) were dissolved in 40 mL of deionized water under stirring for 2 h to activate carboxyl groups. Subsequently, Bis-5HT-Glu-NH 2 (3 mmol) and Dex-NH 2 (3 mmol) were added and continuously stirred for 24 h. The resulting mixture underwent purification via dialysis against deionized water. The final product was obtained through lyophilization, and the chemical structure of PSA-Dex-Bis-5HT was confirmed by 1 HNMR analysis. 2.5. Preparation and characterization of PD5NPs In brief, PSA-Dex-Bis-5HT was dissolved in methanol, and a dry thin film was formed through rotary evaporation [ 19 ]. Subsequently, the thin film was rehydration with deionized water to produce a PD5NPs dispersion. The particle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS) with a Zetasizer Nano instrument (Anton Paar, Austria). The morphology of PD5NPs was visualized through transmission electron microscopy (TEM, JEOL TEM-1011, Japan). The conjugation ratio of Dex in PSA-Dex-Bis-5HT was assessed by high-performance liquid chromatography (HPLC, Agilent 1260, USA) following hydrochloric acid (HCl, 0.1 M) treatment. 2.6. Stability evaluation To assess the in vitro stability of PD5NPs, dynamic light scattering (DLS) was employed to measure particle size and polydispersity index (PDI) over time. The stability in serum was investigated through monitoring the change in particle size and PDI of PD5NPs incubated with rat serum. 2.7. In situ size amplification and characterization of aggregates PD5NPs solution were incubated with PBS containing MPO (at concentrations of 10 U) and 3% H 2 O 2 for 4 h. The PSA-Dex nanoparticles (PDNPs) without Bis-5HT modified was selected as control group. The alterations in size distribution and morphology were monitored using both DLS and TEM. The zeta potential of aggregates was determined by DLS. To evaluate the stability in serum of prepared aggregates, TEM was employed to measure the particle size and morphology over time. 2.8. In vitro release assay To assess the pH-dependent release of Dex, PD5NPs and prepared aggregates were resuspended in PBS with different pH values. Subsequently, each sample was introduced into a dialysis bag and placed in a glass bottle containing release medium with diverse pH conditions. The in vitro release assay was carried out under gentle agitation at 37°C. At specified time intervals, aliquots of the release medium were collected, and the Dex concentration was quantified using HPLC. 2.9. Cell cytotoxicity To determine the cytotoxicity of PD5NPs on human umbilical vein endothelial cells (HUVECs) and Raw264.7, 1×10 4 cell per well were seeded in 96-well plates were treated with 100 µL of PD5NPs and prepared aggregates at final concentrations ranging from 7.81 to 500 µg/mL. After 24 h of incubation, MTT solution (20 µL, 5 mg/mL) was added and incubated for 4 h. DMSO was then added to dissolve the blue formazan crystals. The absorbance of each well was measured using a microplate reader at 490 nm. 2.10. Establishment of arthritis model The Adjuvant-Induced Arthritis (AIA) model was induced in male Sprague-Dawley rats (8–10 weeks) using Complete Freund's Adjuvant (CFA) containing 10 mg/mL of mycobacterium tuberculosis. Specifically, 50 µL of CFA was subcutaneously injected into the hind paw of each Sprague-Dawley rat. The swelling of all rat limbs was monitored every other day post-induction. All animals were maintained under standard conditions with ad libitum access to food and water. 2.11. MPO detection in AIA rats To compare the MPO level between healthy and arthritic rats, MPO concentration in arthritic joints and healthy joints were determined using an ELISA kit. Arthritic rats exhibited observable joint redness and swelling were sacrificed and ankle joint tissues were dissected. The MPO concentration in joint homogenate was measured according to the manufacturer's instructions. 2.12. In vivo biodistribution study To elucidate the in vivo biodistribution of PD5NPs, Cy5.5-labeled formulations were employed for visualized observation using an in vivo imaging system (IVIS, Perkin Elmer, USA). The Cy5.5-labeled PDNPs were chosen as the control group. AIA rats were randomly assigned to three groups and intravenously injected with free Cy5.5, Cy5.5-PDNPs, or Cy5.5-PD5NPs. At specified time points, fluorescence signal in inflamed joints in AIA rats, as well as the fluorescence distribution in major organs and joint tissues were measured by IVIS. 2.13. Co-localization of blood vessel To investigate the colocalization of PD5NPs with hyperplastic vessels in inflamed synovium, rats receiving Cy5.5-PDNPs or Cy5.5-PD5NPs were euthanized at indicated time points, and synovium tissues were collected for preparing frozen sections. The obtained cryosections of synovium samples were fixed by 4% paraformaldehyde for 10 min and blocked with 5% fetal bovine serum. Subsequently, anti-CD31 antibody (Abcam, USA) was incubated with slices at 4°C overnight, followed by IgG-AlexaFluor 488 (Abcam, USA) incubation for 30 min. DAPI was used for nucleus staining. The colocalization of Cy5.5-PD5NPs with CD31-labeled vessels was then observed using a fluorescence microscope. 2.14. Pharmacokinetic behavior of Dex in plasma and inflamed joints AIA rats were randomly allocated into three groups and intravenously administrated with one of the following: free Dex, PDNPs, or PD5NPs at a Dex dosage of 2 mg/kg. At predetermined intervals, rats from each group were euthanized to collect both blood samples and joint tissues. The concentration of Dex in plasma and joint homogenates was quantified using liquid chromatography mass spectrometry (LC-MS/MS, Agilent, USA). Pharmacokinetic parameters, including the area under the concentration-time curve (AUC 0 − t ), half-life (T 1/2 ), and mean retention time (MRT 0 − t ) were analyzed using DAS software. 2.15. Therapeutic efficacy AIA rats were randomly assigned to four groups, with healthy rats serving as the normal control (n = 5). AIA rats received intravenous injection of one of the following: PBS, free Dex, PDNPs, and PD5NPs (at a Dex dose of 2 mg/kg) every other day for three times. During the period of the treatments, the body weight, paw thickness measured via vernier caliper and joint score of the AIA rats were monitored every day in each group. The joint score determined by grading from score of 0 (no observable erythema or swelling) to 4 (severe swelling and erythema) was given for each paw. The swelling and ulceration degree of 4 paws were scored, finally resulting in a maximum possible score of 16 for each animal [ 26 ]. After a two-week period, rats were euthanized and joints tissues were collected for subsequent pathological examination. The joint tissues from each group were cut up and homogenized. The concentration of inflammatory cytokines (TNF-α and IL-1β) in joint homogenates was measured by ELISA kits. The joint ankles from each group were soaked in 4% paraformaldehyde solution for three days and then incubated in 15% EDTA-2Na decalcification solution until the samples could be easily sliced. The joint slices were finally stained with hematoxylin and eosin to observe the inflammatory cell infiltration and cartilage erosion. For toluidine blue staining and safranin solid green staining, slices were incubated with specific reagent and observed via microscopy to analyze the cartilage integrity and tissue damage. 2.16. The colocalization of lymphatic vessels and PD5NPs AIA rats were randomly allocated into three groups and were administrated intravenously one of the following: free Cy5.5, Cy5-PDNPs, or Cy5.5-PD5NPs. At predetermined time points, rats were euthanized, and synovium tissues were collected. Cryosections of synovium tissues were sequentially incubated with anti-LYVE-1 antibody (Abcam, USA) and IgG-Alexa Fluor 488 for specific labeling of lymphatic vessels. DAPI was added for nucleus staining. All samples were examined using a fluorescence microscope. Colocalization analysis was conducted using Image J software. 2.17. Synovium interstitial lymphatic inhibition Interstitial lymphatic growth in inflamed synovium was inhibited using the small molecule MAZ51. MAZ51 dissolved in DMSO (100 µg/mL) was loaded into a 29-gauge insulin syringe and intra-articular administered to joints. An intra-articular injection of the MAZ51(5 µL) ensures all drug molecules are within the target site while minimizing off-target effects. Meanwhile, AIA rats with intra-articular injection of PBS were selected as control. This intra-articular injection of MAZ51 was repeated daily for five days. PDNPs and PD5NPs were intra-articular administered on the sixth day. Ankle joint with the surrounding tissues and lymph nodes (popliteal and inguinal lymph nodes) were resected after 4 h and homogenized for LC-MS/MS detection. 2.18. In vivo safety evaluation The body weight of each rat was monitored throughout the treatment period. Rats were euthanized to collect blood samples for blood routine test and biochemical analysis, encompassing assessments of alanine transaminase (ALT), aspartate aminotransferase (AST), as well as blood urea nitrogen (BUN) and creatinine (Cr) levels. Additionally, major organs were harvested and subjected to hematoxylin and eosin (H&E) staining for histopathological examination. 2.19. Statistical analysis All data were presented as mean ± standard deviation (SD) of minimum three replicates as indicated. Comparisons among multiple groups were expressed by one-way ANOVA. The significant difference between two comparative groups was analyzed by student’s t-test. All statistical analysis were performed using Graphpad Prism 7.0, P value of < 0.05 was considered as significant difference. 3. Results and Discussion 3.1. Synthesis and characterization of PD5NPs and aggregates The synthesis of PSA-Dex-Bis-5HT involved a three-step process as shown in Figure S1 -2. Successful synthesis of Bis-5HT-Glu-NH 2 was evidenced by the disappearance of characteristic peaks at 4.29–4.2 ppm (m, 2H) and 4.01–3.93 ppm (m, 1H) in the 1 H NMR spectrum (Figures S3). The 1 HNMR spectrum of PSA-Dex-Bis-5HT exhibited distinctive signals at 10.47 ppm (s, 2H), 8.02–7.98 ppm (m, 1H), and 7.34–7.44 ppm (m, 1H), which were absent in the spectrum of PSA alone (Figures S4). These findings demonstrated the successful synthesis of PSA-Dex-Bis-5HT. It was hypothesized that in the presence of MPO and hydrogen peroxide (H 2 O 2 ), Bis-5HT on the surface of PD5NPs can undergo oxidation and radical generation, which can subsequently engage in particle oligomerization and binding to phenolic residues from the proteins (Fig. 2 A). Firstly, PD5NPs were prepared using a thin film hydration method [ 27 ]. The average size and PDI of prepared PD5NPs exhibited minimal changes even after one week, indicating robust stability under static condition (Fig. 2 B). When exposed to plasma condition at room temperature, no significant change in particle size or PDI were observed throughout the observation period (Fig. 2 C), suggesting favorable stability of PD5NPs in serum. As shown in Fig. 2 D, PD5NPs exhibited a particle size approximately 150 nm with a narrow distribution, as determined by dynamic light scattering (DLS) at the initial time. After MPO and H 2 O 2 treatment for 4 h, the particle size of PD5NPs significantly increased and the size distribution become wide and disordered (Fig. 2 E). In accordance with DLS measurement, TEM images of PD5NPs revealed a uniform and near-spherical morphology in the absence of MPO and H 2 O 2 (Fig. 2 F). However, as shown in Fig. 2 G and Figure S5, obvious and widespread particle aggregation could be seen in different fields. Moreover, the zeta potential of the aggregates was about − 22.21 ± 0.08 mV measured using DLS. When incubated in serum, no significant morphology change of aggregates was observed throughout the observation period according to TEM results, suggesting favorable stability of these aggregates in serum (Figure S6). TEM images showed that the size of aggregates in serum was approximate range from 500 nm to 3 µm. In our study, PDNPs without Bis-5HT modification was used as a control. PDNPs showed similar particle size as PD5NPs (Figure S7). When incubated with MPO and hydrogen peroxide for 4 h and 24 h, the particle size of PDNPs remained no significant change (Figure S8). In contrast, the size distribution of PD5NPs underwent a marked increase after incubation with MPO and hydrogen peroxide for 4 h via DLS (Fig. 2 E) and TEM (Fig. 2 G). These results indicated that Bis-5HT is necessary for aggregation. The Dex conjugation ratio to PD5NPs was calculated to be approximately 7.5%, as assessed by HPLC. In Fig. 2 H and Figure S9, in vitro release studies revealed that both PD5NPs and aggregates displayed a slow and sustained release profile in PBS at pH 7.4, whereas Dex release was notably accelerated under acidic pH condition. Furthermore, both Raw264.7 and HUVEC cell lines exhibited favorable cell viability when incubated with PD5NPs and aggregates over a concentration range of 7.81 to 500.00 µg/mL (Fig. 2 I-J and Figure S10). Hence, it can be concluded that PD5NPs and aggregates demonstrate excellent cytocompatibility. 3.2. Inflammation targeting behavior of PD5NPs To assess the targeting ability of PD5NPs to arthritic joints, Cy5.5-PD5NPs were administered intravenously to adjuvant-induced arthritis (AIA) rats, and the biodistribution of PD5NPs was monitored using an in vivo imaging system (IVIS). The Cy5.5-labled PSA-Dex nanoparticles (PDNPs) without 5-HT modification were chosen as the control group. In Fig. 3 A and Figure S11, the fluorescent signal in the inflamed joints of rats receiving free Cy5.5 was barely detectable, with predominant fluorescence observed in the liver and kidney. In contrast, intensive fluorescent signal was observed at arthritic sites in rats treated with Cy5.5-PD5NPs and Cy5.5-PDNPs at 2 h post-injection. Nevertheless, fluorescent signal in rats receiving Cy5.5-PD5NPs remained remarkable even after 24 h, while rats receiving Cy5.5-PDNPs displayed very limited signal. The fluorescent quantitative analysis provided more direct comparison. The fluorescent intensity of joints in Cy5.5-PD5NPs group is nearly 2-fold higher than that in PDNPs group at 24 h post-injection (Fig. 3 B). This considerable difference in fluorescent intensity at 24 h between Cy5.5-PD5NPs and Cy5.5-PDNPs might be due to the delayed lymphatic transportation endowed by in-situ size amplification of PD5NPs. Figure 3 C further corroborated this assumption. The arthritic joints from AIA rats exhibited a substantial increase in MPO expression compared to that from normal rats (Fig. 3 C). Furthermore, the more detailed colocalization of Cy5.5-PD5NPs and blood vessel within inflamed tissues was visualized in Fig. 3 D. Consistent with IVIS findings, both PDNPs and PD5NPs treated groups displayed more robust fluorescence compared to free Cy5.5 group during our observation period (Fig. 3 E). At 6 h and 24 h, a significant overlap between red and green fluorescence indicated that both PDNPs and PD5NPs reached inflamed synovium through local hyperplastic vessels (Fig. 3 F-G). Both the Pearson’s coefficient for Cy5.5-PDNPs and Cy5.5-PD5NPs remained around 0.8 at 6 and 24 h, indicating a high degree of overlap with hyperplastic vessels (Figure S12). However, at 24 h, the colocalization areas of red and green fluorescence diminished, suggesting drug formulations gradually exited from inflamed synovium. Notably, the PD5NPs treated group exhibited much stronger red fluorescence than the PDNPs group at 24 hours after injection (Fig. 3 E). We supposed that the in-situ size amplification of PD5NPs in response to MPO and H 2 O 2 can significantly enhance drug retention in arthritic sites. 3.3. In vivo pharmacokinetics behavior of PD5NPs To further investigate the in vivo behaviors including pharmacokinetic and synovium retention of PD5NPs in arthritic joints, more accurate in vivo quantitative analysis was performed (Fig. 4 A). The plasma pharmacokinetic curve was shown in Fig. 4 B. The calculated area under the curve (AUC 0 − t ) and half-time (T 1/2 ) of PDNPs and PD5NPs significantly increased compared to free Dex group (Fig. 4 C-D), while the difference in MRT 0 − t between PDNPs and PD5NPs was not obvious in plasma (Fig. 4 E). These results suggested they can similarly enhance the Dex bioavailability and extend the in vivo circulation time. However, a notable contrast in pharmacokinetic behavior emerged in inflamed joints (Fig. 4 F). Despite rapid decline in Dex concentration over time in both groups, PD5NPs exhibited a Dex concentration of nearly 12 ng/mL within inflamed joints after 36 h post administration, while only 2 ng/mL was observed in PDNPs groups. Furthermore, PD5NPs demonstrated a significant increase in AUC 0 − t in joints compared to the PDNPs groups (Fig. 4 G). The T 1/2 of Dex in inflamed joints in PD5NPs treated group was nearly up to 10 h, which was much higher than that in the PDNPs group (Fig. 4 H). The MRT 0 − t of the PD5NPs group was almost 2-fold higher than that in PDNPs treated group, indicating the significantly prolonged drug retention within arthritic joints after PD5NPs injection (Fig. 4 I). These might be due to the in-situ size amplification of PD5NPs from small nanoparticles to larger aggregates in response to MPO and H 2 O 2 . 3.4. In vivo lymphatic transport Nanoparticles typically exhibit directional movement from areas of higher pressure to those of lower pressure. Microvascular pressure falls within the range of 2–40 mmHg [ 28 ], while the internal pressure of lymphatic vessels is quite lower, generally about − 2 mmHg [ 29 ]. This creates a pressure gradient that predominantly guides the movement of nanoparticles toward lymphatic capillaries. Additionally, lymphatic vessels are distinguished from blood vessels due to the presence of specialized ultrastructural attributes including channel networks and a series of interconnected vesicles [ 30 ], which are responsible for trans-endothelial transport of macromolecule. Therefore, we can infer that PDNPs and PD5NPs might enter inflamed sites through blood capillaries but are probably transported out of synovium via lymphatic capillaries. To figure out whether PD5NPs can delay the lymphatic clearance, we explored the colocalization of lymphatic vessels with Cy5.5-PD5NPs after injecting Cy5.5-PD5NPs to arthritic rats as shown in Fig. 5 A. Figure 5 B illustrates a rapid decrease in free Cy5.5 signal, with fluorescence becoming undetectable within 24 h. Both Cy5.5-PDNPs and Cy5.5-PD5NPs significantly prolonged drug retention within inflamed joints, with PD5NPs exhibiting a substantially higher fluorescence intensity than PDNPs at the 24 h (Figure S13). The colocalization degree of lymphatic vessels and Cy5.5-PDNPs was notably more pronounced than that of Cy5.5-PD5NPs, suggesting higher distribution of Cy5.5-PDNPs within lymphatic tissues. Additional colocalization analysis of red and green fluorescence at 24 h further revealed that PD5NPs had less colocalization with lymphatic vessels than PDNPs (Fig. 5 C-D). Pearson’s coefficient, which reflects the degree of colocalization between the red and green signals [ 31 ], also confirmed this observation. As illustrated in Fig. 5 E, the Pearson’s coefficient for Cy5.5-PDNPs remained around 0.8 at both 6 and 24 h, indicating a high degree of overlap with lymphatic vessels. Conversely, the Pearson’s coefficient for Cy5.5-PD5NPs was approximately 0.2 at 24 h, denoting minimal distribution in lymphatic vessel. These results collectively verified that PD5NPs were able to restrain the lymphatic transport and delay the lymphatic clearance compared with PDNPs. In general, nanoparticles in inflamed sites can be transported to lymphatic system via paracellular transport and transcellular transport [ 32 ]. The pressure gradient between microvasculature and lymphatic vessels, and the existence of lymphatic endothelial gap, usually contribute to the paracellular transport of nanoscale particles from interstitial space to lymph nodes [ 33 ]. Vascular endothelial growth factor receptor 3 (VEGFR3) is predominantly expressed on lymphatic endothelial cells and plays a key role in lymph angiogenesis [ 34 ]. The small molecule MAZ51 has been demonstrated to effectively disrupt lymph angiogenesis-driven pathology by antagonizing VEGFR-3 signaling [ 35 ], thereby interfering the lymph angiogenesis in synovium. The proposed transport pathway of PDNPs and PD5NPs via lymphatic vessels were shown in Fig. 6 A. To further elucidate the transport ability of PD5NPs to lymph vessels in inflamed joints, we conducted a comparative analysis of drug transport from synovium to lymphatic systems by administering the joint with and without the VEGFR3-specific inhibitor MAZ51 (Fig. 6 B). As shown in Fig. 6 C and 6 E, the MAZ51 treatment resulted in significantly elevated PDNPs retention in inflamed joints and decreased accumulation in lymph nodes, suggesting that PDNPs might be mainly cleared from synovium through angiogenesis of lymph vessels. Moreover, MAZ51 treatment had no impact on the PD5NPs retention in inflamed joints (Fig. 6 D), further implying that the transport process of PD5NPs from inflamed synovium to lymphatic vessels was almost inhibited even in the absence of MAZ51. Compared with PBS treatment, MAZ51 can inhibit lymph angiogenesis and slow down the rate of lymphatic vessels-dependent transport. The lymphatic transport of large-sized aggregates is relatively reduced compared with smaller particles. And the MAZ51-mediated shrinkage of lymphatic vessels might further interfere the transportation of PD5NPs aggregates towards lymphatic system (Fig. 6 F). Collectively, based on these findings, we can speculate that the clearance of PDNPs mainly depends on the lymphatic transport, while PD5NPs appeared to greatly inhibit the lymphatic clearance via in-situ size amplification. 3.5. Therapeutic efficacy The therapeutic efficacy of PD5NPs was investigated in an AIA rat model and the treatment regimen was outlined in Fig. 7 A. The AIA model was successfully established and AIA rats were randomly allocated into four groups and administered PBS, free Dex, PDNPs, or PD5NPs at a Dex dosage of 2 mg/kg on days 8, 10, and 12, respectively. As depicted in Fig. 7 B, the hind paws of rats in PBS treated group exhibited pronounced swelling and ulceration. Treatment with PDNPs resulted in a modest reduction in swelling, whereas PD5NPs treatment significantly mitigated joint swelling, restoring the joints to nearly normal condition. The progression of joint scores and paw thickness among all groups corresponded closely with the visual assessment of joint morphology, as illustrated in Fig. 7 C-D. Compared to PBS group, free Dex treatment achieved a marginal decrease in joint score and paw thickness. In contrast, PD5NPs effectively abrogated joint swelling and symptoms of RA. The area under the curve (AUC) analysis for joint score (Fig. 7 E) and paw thickness (Fig. 7 F) showed a direct and quantitative comparison among these treatment groups, corroborating the abovementioned observation. Furthermore, the levels of pro-inflammatory cytokines in arthritic joints, tumor necrosis factor-alpha (TNF-α) (Fig. 7 G), and interleukin-1 beta (IL-1β) (Fig. 7 H), were significantly elevated in PBS treated group of AIA rats. Conversely, PD5NPs treatment exhibited the lowest levels of TNF-α and IL-1β among all groups, underscoring the potent anti-inflammatory property of PD5NPs. To elucidate the histopathological changes after various therapeutic interventions, the ankle joints from all experimental rats were sectioned and subjected to microscopic examination. Hematoxylin and eosin (H&E) staining of the articular tissues from PBS treated group disclosed a marked constriction of the joint space, accompanied by pervasive inflammatory cell infiltration and pronounced cartilage degradation (Fig. 7 I). Conversely, PD5NPs treated group exhibited a remarkable restoration of the articular space, negligible inflammatory infiltration, and only minimal sign of cartilage damage. For a more detailed assessment of cartilage integrity and tissue damage within the joint tissues, toluidine blue staining was utilized [ 36 ]. Figure 7 I (middle) revealed significant cartilage deterioration and fibrotic overgrowth in PBS treated group, indicating serious articular damage. In contrast, PD5NPs treatment markedly attenuated cartilage erosion. Additionally, the integrity of the cartilaginous matrix was evaluated using safranine O and fast green (SO&FG) staining [ 37 ]. PBS group displayed a paucity of red staining, reflecting substantial cartilage depletion and osseous tissue necrosis. In comparison, the PD5NPs group manifested an extensive red staining area, denoting robust cartilage preservation and potential reparative activity. Collectively, these results demonstrated that PD5NPs not only ameliorate synovial inflammation but also significantly inhibit cartilage degradation in the AIA rat model. 3.6. In vivo biocompatibility evaluation To further elucidate the in vivo biocompatibility of PD5NPs, rats were sacrificed for a safety evaluation (Fig. 8 A). Throughout the treatment regimen, rats in all groups exhibited continuous body weight gain (Fig. 8 B). Furthermore, there was no difference among all groups in terms of red blood cell (RBC), white blood cell (WBC) and platelet (PLT) level (Fig. 8 C-E), suggesting good compatibility of our treatment. Likewise, there was no significant variance observed in the serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) among all groups (Fig. 8 F-I), indicating that our treatment did not induce liver or renal damage. Additionally, the H&E staining showed that no inflammation infiltration or cellular necrosis was detected in major organs among all groups (Figure S14). Hence, our treatment demonstrated good biocompatibility. 4. Conclusion In summary, we developed an in-situ size amplification strategy with the objective of suppressing in vivo lymphatic clearance and extending the therapeutic efficacy in RA treatment. The prepared PD5NPs can transform from small-sized nanoparticles to larger-sized aggregates in the presence of MPO and H 2 O 2 in inflamed sites. Following intravenous administration, PD5NPs exhibited prolonged blood circulation in vivo and increased retention at inflamed joints. Notably, PD5NPs effectively impeded drug clearance through lymphatic system by undergoing in-situ size amplification into aggregates in response to inflammatory condition. In the arthritic model, PD5NPs efficiently attenuated RA development and promoted cartilage tissue repairing. Altogether, this in situ size amplification strategy exhibited great potential to enhance the therapeutic efficacy in RA treatment. Declarations Ethics approval and consent to participate All animal studies were approved by the Ethics committee of Sichuan Provincial People's Hospital before starting any animal experiments. All experiments were conducted following the guidelines of the National Institute of Health for the ethical care and handling of laboratory animals. Consent for publication All authors concur with the submission and publication of this paper. Appendix A. Supporting information Supporting data to this article can be found online at xxx. Conflicts of interest The authors declare no competing financial interest. Funding This study was supported by the National Natural Science Foundation of China (No. 82404541, No. 82003661 and No.82271120), Postdoctoral Fellowship Program of CPSF under Grant Number GZC20240218, Postdoctoral Fund of Sichuan Provincial People's Hospital (No.2023BH10), Natural Science Foundation of Sichuan Province of China (No. 2023NSFSC1679), Sichuan Science and Technology Program (No. 2022ZYD0131), the CAMS Innovation Fund for Medical Sciences (No.2019-I2M-5-032), and the Department of Science and Technology of Sichuan Province, China (No.2024ZHYS0018). Author contributions Yi Shi and Qin Wang conceived and supervised the research. Xianyan Qin and Luhan Zhang carried out the experiments and performed data analysis. Yang-Bao Miao and Linxi Jiang participated in part of the experiments. Liang Zou provided intellectual discussions on experimental designs. Xianyan Qin wrote the manuscript. Qin Wang, Yang-Bao Miao and Yi Shi revised the manuscript. All authors have read and approved the final manuscript. References Deane KD, Holers VM, Rheumatoid Arthritis Pathogenesis, Prediction, and Prevention: An Emerging Paradigm Shift. Arthritis Rheumatol. 2021;73:181-93. Komatsu N, Takayanagi H, Mechanisms of joint destruction in rheumatoid arthritis - immune cell-fibroblast-bone interactions. Nat. Rev. Rheumatol. 2022; 18:415-29. Fang RH, Zhang L, Biohybrid nanoparticles for treating arthritis. Nat Nanotechnol 2023; 18:1387-8. Nooreen R, Nene S, Jain H, Prasannanjaneyulu V, Chitlangya P, Otavi S, Khatri DK, Raghuvanshi RS, Singh SB, Srivastava S, Polymer nanotherapeutics: A versatile platform for effective rheumatoid arthritis therapy. J. Control. Release 2022;348:397-419. Zhang A, Meng K, Y Liu, Pan Y, Qu W, Chen D, Xie S, Absorption, distribution, metabolism, and excretion of nanocarriers in vivo and their influences. Adv. Colloid. Interface Sci. 2020; 284:102261-9. Wang Q, Qin XY, Fang JY, Sun X, Nanomedicines for the treatment of rheumatoid arthritis: State of art and potential therapeutic strategies. Acta pharmaceutica Sinica. B 2021; 11:1158-74. Koch AE, The role of angiogenesis in rheumatoid arthritis: recent developments. Ann. Rheum. Dis. 2000; 59:65-71. Bouta EM, Bell RD, Rahimi H, Xing L, Wood RW, Bingham CO, Ritchlin CT, Schwarz EM, Targeting lymphatic function as a novel therapeutic intervention for rheumatoid arthritis. Nat. Rev. Rheumatol. 2018;14:94-106. Evans CH, Kraus VB, Setton LA, Progress in intra-articular therapy. Nat. Rev. Rheumatol. 2014; 10:11-22. DiDomenico CD, Lintz M, Bonassar LJ, Molecular transport in articular cartilage - what have we learned from the past 50 years? Nat. Rev. Rheumatol. 2018;14:393-403. Li X, Dai B, Guo J, Zheng L, Guo Q, Peng J, Xu J, Qin L, Nanoparticle-Cartilage Interaction: Pathology-Based Intra-articular Drug Delivery for Osteoarthritis Therapy, Nano-Micro Lett. 2021;13:149-97. 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Guyton AC, Granger HJ, Taylor AE, Interstitial fluid pressure. Physiol. Rev. 1971;51:527-63. Petrova TV, Koh GY, Biological functions of lymphatic vessels. Science 2020;369:eaax4063. Adler J, Parmryd I, Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient. Cytom. Part. A 2010; 77:733-742. McCright J, Yarmovsky J, Maisel K, Para- and Transcellular Transport Kinetics of Nanoparticles across Lymphatic Endothelial Cells. Mol. Pharmaceut. 2023; 21:1160-9. Tang Y, Liu B, Zhang Y, Liu Y, Huang Y, Fan W, Interactions between nanoparticles and lymphatic systems: Mechanisms and applications in drug delivery. Adv. Drug Deliv. Rev. 2024;9:115304-24. Zhang Y, Ulvmar MH, Stanczuk L, Martinez-Corral I, Frye M, Alitalo K, Mäkinen T, Heterogeneity in VEGFR3 levels drives lymphatic vessel hyperplasia through cell-autonomous and non-cell-autonomous mechanisms. Nat. Commun. 2018; 9:1296-302. Yamamura A, Nayeem MJ, Muramatsu H, Nakamura K, Sato M, MAZ51 Blocks the Tumor Growth of Prostate Cancer by Inhibiting Vascular Endothelial Growth Factor Receptor 3. Front. Pharmacol. 2021; 20:667474-86. Schmitz N, Laverty S, Kraus VB, Aigner T, Basic methods in histopathology of joint tissues. Osteoarthr. Cartilage 2010; 18:113-6. Rutgers M, Pelt MJP, Dhert WJA, Creemers LB, Saris DBF, Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage. Osteoarthr. Cartilage 2010; 18:12-23. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Graphicalabstractimage.png Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 21 Oct, 2024 Reviews received at journal 09 Oct, 2024 Reviews received at journal 09 Oct, 2024 Reviews received at journal 08 Oct, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers agreed at journal 17 Sep, 2024 Reviewers agreed at journal 17 Sep, 2024 Reviewers invited by journal 17 Sep, 2024 Editor assigned by journal 13 Sep, 2024 Submission checks completed at journal 13 Sep, 2024 First submitted to journal 11 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5069556","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":368812765,"identity":"253e688f-0bde-45aa-ae2e-c13dfa064757","order_by":0,"name":"Xianyan Qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDACZhBhICHHz8x8+AEJWgosjCXb2dIMSLDqQ0XihvM8ChJEKeY7zntMmsdAwtj4MA+DAUONTTRBLZKH+ZKNgVrkzA7zHnjAcCwtt4GQFoPDPIaPQbaYHeZLMGBsOEyUFhCSSNzcDCSJ1QK2JXEDM7FaJA/zGBvOATpM4jAwkBOI8Qvf+TNmEm/+1Mnx9x8+/OBDjQ1hLQwHGBiYeGCcBILKoVoYfxClchSMglEwCkYsAAD20jkaZ3VCxQAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xianyan","middleName":"","lastName":"Qin","suffix":""},{"id":368812766,"identity":"d2799033-74ab-49e2-94ed-bd06ec87153b","order_by":1,"name":"Luhan Zhang","email":"","orcid":"","institution":"Sichuan Academy of Medical Sciences, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Luhan","middleName":"","lastName":"Zhang","suffix":""},{"id":368812768,"identity":"f001f71a-0c9c-4e5d-8aa7-87417eb70725","order_by":2,"name":"Yang-Bao Miao","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang-Bao","middleName":"","lastName":"Miao","suffix":""},{"id":368812769,"identity":"a81db915-7a0d-44e5-8209-9a5d87466f96","order_by":3,"name":"Linxi Jiang","email":"","orcid":"","institution":"Sichuan Academy of Medical Sciences, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Linxi","middleName":"","lastName":"Jiang","suffix":""},{"id":368812770,"identity":"5a6c65e2-22f5-40ad-952c-fb990c8df725","order_by":4,"name":"Liang Zou","email":"","orcid":"","institution":"Chengdu University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zou","suffix":""},{"id":368812772,"identity":"be57b041-8207-4d71-8a8c-a3c24427ea66","order_by":5,"name":"Qin Wang","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Wang","suffix":""},{"id":368812774,"identity":"6878aa3e-83c1-40fb-a7fc-2290e80cb836","order_by":6,"name":"Yi Shi","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-09-11 08:45:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5069556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5069556/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-03061-8","type":"published","date":"2024-12-18T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68227714,"identity":"ceac00a1-de17-49a7-ae5a-bc0e9f649185","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":425422,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of the formation of nanoparticles and aggregates. (B)The \u003cem\u003ein vivo\u003c/em\u003e behavior and transport of PD5NPs in arthritic rats.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/ea77ab24e3449993c9a7d5f7.png"},{"id":68227720,"identity":"d95d4afa-ecb2-4a8e-b912-624a628c11bc","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270430,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of the aggreagtion triggered by Bis-5HT-Glu-NH\u003csub\u003e2\u003c/sub\u003e in respond to MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (B) The stability of PD5NPs at room temperature. (C) The stability of PD5NPs incubated with serum at room temperature. Size distribution of PD5NPs incubated in MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution at 0 h (D) and 4 h (E), respectively. Colored lines represented three parallel samples. Representative TEM images of PD5NPs incubated in MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution at 0 h (F) and 4 h (G), respectively. Scale bar = 0.5 µm. (H) \u003cem\u003eIn vitro\u003c/em\u003e release behavior of Dex from PD5NPs in PBS buffer with different pH values. Viability of Raw264.7 (I) and HUVEC (J) cells after 24 h incubation with PD5NPs at different concentrations. Results were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/54a3d745f519f4933027491f.png"},{"id":68227713,"identity":"87f19b8f-6136-466b-939a-3d44196c180b","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":562446,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative biodistribution images of arthritic rats after the intravenous injection of free Cy5.5, Cy5.5-labled PDNPs and Cy5.5-labled PD5NPs at 2 h, 6 h and 24 h. (B) Fluorescence semi-quantitative results in joints after the intravenous injection of free Cy5.5, Cy5.5-labled PDNPs and Cy5.5-labled PD5NPs at 24 h. (C) The concentration of MPO in the joints of normal rats and AIA rats. (D) Represent fluorescent images of synovium tissue treated with free Cy5.5, Cy5.5-PDNPs and Cy5.5-DKPNPs at 6 h and 24 h. DAPI, blue; CD 31, green; Cy5.5, red. Scale bar = 100 μm. Results were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001. (E) Semi-quantification of the fluorescence signal after treating with Cy5.5-PDNPs and Cy5.5-PD5NPs at 24 h. The quantitative analysis of colocalization between Cy5.5 (red) and CD31 (green) after treating with Cy5.5-PDNPs (F) and Cy5.5-PD5NPs (G) after 24 h.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/904cf27a03b382af6019fce8.png"},{"id":68227865,"identity":"6b0c806c-35cc-4f90-adf3-e40ee5e3ccbe","added_by":"auto","created_at":"2024-11-05 04:33:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":184523,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of pharmacokinetic study after intravenous injection. (B) Pharmacokinetics profiles of Dex in plasma after the intravenous injection of free Dex, PDNPs and PD5NPs at different time points. The AUC0-t (C), T\u003csub\u003e1/2\u003c/sub\u003e (D) and MRT\u003csub\u003e0-t \u003c/sub\u003e(E) of Dex in plasma after treatment of free Dex, PDNPs and PD5NPs. (F) Pharmacokinetics profiles of Dex in joints after the intravenous injection of free Dex, PDNPs and PD5NPs at different time points. The AUC0-t (G), T\u003csub\u003e1/2\u003c/sub\u003e (H) and MRT\u003csub\u003e0-t \u003c/sub\u003e(I) of Dex in plasma after treatment of free Dex, PDNPs and PD5NPs. Results were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=4. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001, ns: not significant, p\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/6229fecc2864904874898a81.png"},{"id":68228315,"identity":"4a93c4ac-7253-4b64-b822-073e14ed9cce","added_by":"auto","created_at":"2024-11-05 04:41:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":487078,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of experimental design. (B) Represent fluorescent images of synovium treated free Cy5.5, Cy5.5-PDNPs and Cy5.5-PD5NPs at 6 h and 24 h. DAPI, blue; LYVE-1, green; Cy5, red. Scale bar=100 μm. The quantitative analysis of colocalization between Cy5.5 (red) and LYVE-1 (green) after treating with Cy5.5-PDNPs (C) and Cy5.5-PD5NPs (D) after 24 h. (E) The Pearson’s coefficient of colocalization between Cy5.5 (red) and LYVE-1 (green) after treating with Cy5.5-PDNPs and Cy5.5-PD5NPs at 6 h and 24 h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/1a1604a669909622a185b73c.png"},{"id":68227718,"identity":"5f775a76-e106-4e5d-97bc-35f601f2cc40","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":225225,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of the exit pathway of PDNPs and PD5NPs via lymphatic system. (B) The experimental design of synovium lymphatics inhibition by intra-articular injection of small molecule MAZ51. The concentration of Dex in joint after PDNPs (C) or PD5NPs treatment (D). The concentration of Dex in lymph node after PDNPs (E) or PD5NPs treatment (F). Results were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=4. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001, ns: not significant, p\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/90626c223e9d350578e5b9bc.png"},{"id":68227722,"identity":"f6f165cf-8788-445f-b9f8-c12252c4c6dc","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":842438,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic efficacy of PD5NPs in SD rats with arthritis. (A) Therapeutic schedule for the treatment of arthritic rats. (B) Representative images of the hind paws after the treatment with PBS, free Dex, PDNPs and PD5NPs, respectively. The joint score (C) and paw thickness (D) after the treatment of PBS, free Dex, PDNPs and DKPNPs. The AUC score of joint score (E) and paw thickness (F) after the treatment of PBS, free Dex, PDNPs and PD5NPs. The inflammatory levels of TNF-α (G) and IL-1β (H) in arthritic joints after the treatment with PBS, free Dex, PDNPs and PD5NPs. All results were presented as mean ± SD, \u003cem\u003en\u003c/em\u003e=5. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001, ****p\u0026lt; 0.0001. (I) Representative photographs of H\u0026amp;E staining of ankle joints, Toluidine blue and Safranin O \u0026amp; Fast Green staining (SO\u0026amp;FG) after rats receiving different treatments. scale bar =400 µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/3c4eb4ff1a23211865481c04.png"},{"id":68227723,"identity":"9050a90d-eb2c-4130-8f58-07795e4ad450","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":145528,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Illustration of biocompatibility study in AIA rats. (B) The body weight of arthritis rats in each treatment group. Platelet (C), WBC (D) and (E) RBC counts in blood after the treatment of PBS, free Dex, PDNPs and PD5NPs. The levels of AST (F), ALT (G), Cr (H) and BUN (I) in plasma after the treatment of PBS, free Dex, PDNPs and PD5NPs. Normal rats served as the control group. Results were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=5.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/2c3cf695c59653661aea07f8.png"},{"id":72201711,"identity":"580ccb7f-ff4e-4f4b-81a1-e62fdc999711","added_by":"auto","created_at":"2024-12-23 16:10:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4025237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/1d4408a6-b66a-48ae-824c-5249e893dc10.pdf"},{"id":68227721,"identity":"2feceb7d-d559-4f53-9d31-d11d840a41d7","added_by":"auto","created_at":"2024-11-05 04:25:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8203942,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/8bf536c80948ab324aa550cf.docx"},{"id":68228314,"identity":"b90e0f09-3bc6-42ea-af31-655c292cd3a1","added_by":"auto","created_at":"2024-11-05 04:41:45","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1309362,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstractimage.png","url":"https://assets-eu.researchsquare.com/files/rs-5069556/v1/aa3ffbad77cc0470eac5ce69.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Situ Size Amplification Strategy Suppresses Lymphatic Clearance for Enhanced Arthritis Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs a chronic and uncurable autoimmune disease, rheumatoid arthritis (RA) often requires long-term or even lifelong medication [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Nanomedicines have been widely recognized to enhance delivery efficiency to inflamed sites and improve therapeutic index, thereby reducing administration frequency [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As we known, when administrated in vivo, drug formulations will go through absorption, distribution, metabolism and excretion processes, which play a crucial role on their in vivo safety and effectiveness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Currently, most of the attention have been focused on enhancing targeted distribution in inflamed sites when developing nanomedicine for treating chronic inflammatory diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], very less on slowing down the clearance rate of nanomedicines. In fact, the increased lymph flow and excessive lymph angiogenesis in arthritic sites driven by overactivated immune response will lead to accelerated clearance of nanomedicines from lymphatic system [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To prolong therapeutic efficacy and reduce frequency of drug administration in RA treatment, it is necessary to simultaneously achieve targeted delivery and delayed drug clearance in inflamed sites.\u003c/p\u003e \u003cp\u003eThe joint-space residence time of nanoparticles is heavily influenced by their clearance rate from lymphatic system in synovium [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The particle size plays a pivotal role on the transport and clearance process from inflamed synovium to lymphatic system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Upon systemic administration, nanoparticles enter the joint space via the highly vascularized capillary network of the inflamed sub-synovium and they are primarily cleared from the joint space via the lymphatic system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Generally, nanoparticles smaller than 10 nm are swiftly transported to the blood capillaries, while those within the range of 10\u0026ndash;100 nm effectively distribute in lymphatic capillaries [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Larger nanoparticles might encounter bigger steric hindrance within the gel-like extracellular matrix of joint tissues and exhibit much slower diffusion rate towards lymphatic vessels [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Conversely, nanoparticles with larger sizes often exhibit poor in vivo circulation and rapid phagocytosis by the reticuloendothelial system (RES) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To address this dilemma, an in situ size amplification drug carrier that maintains a small particle size in normal physiological condition while undergoes site-specific size amplification upon reaching inflamed sites, can undoubtedly fulfill the diverse therapeutic demand. Recently, the Xu group designed and synthesized a self-expanding nanogel with multicompartment structure, which was stable in physiological environment while became larger under acidic pH and redox condition, thereby attenuating the side effects and boosting synergistic anticancer effect [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although these nanocarrier system were effective in cancer therapy, it might not be directly utilized to inflammatory conditions. It is worth noting that nanocarriers used in chronic inflammatory diseases should have good biocompatibility and not cause any immune activation or inflammatory responses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our previous work, we designed a transformable nanoparticle which underwent shape transformation from nanoparticles to nanofibers under acidic pH and ligand-receptor interaction, enabling prolonged retention in inflammatory joints [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the re-assembly of KLVFF peptide in this transformable nanoparticle was very susceptible to external mechanical stimuli such as the shear stress in blood flow, posing unpredictable risk during in vivo circulation. Therefore, nanocarriers can undergo size amplification in response to inflammatory stimuli with high specificity will be an efficient and safe strategy.\u003c/p\u003e \u003cp\u003eIn the RA microenvironment, an abundance of neutrophils is recruited to drive the RA progression by secreting a large number of inflammatory cytokines, chemokines and enzymes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], among which myeloperoxidase (MPO) is an azurophilic granule enzyme abundantly expressed by neutrophils [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. MPO can catalyze the conversion of phenolic residues into free radical, contributing to inflammatory response and tissue damage in RA [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to the high level of MPO in inflamed joints and its catalytic characteristic, we proposed an MPO-responsive in-situ size amplification strategy aimed at impeding drug clearance from lymphatic system to ultimately extend therapeutic efficacy. Herein, we synthesized a conjugate consisted of anti-inflammatory dexamethasone (Dex) and naturally occurred polysialic acid (PSA) via an acid-sensitive linker, and modified bis-5-hydroxytryptamine (Bis-5HT) moieties on the PSA backbone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The formed conjugates, termed PSA-Dex-Bis-5HT, can self-assemble into stable nanoparticles (PD5NPs) in physiological condition and facilitate in vivo circulation and targeted delivery to inflamed sites. When reaching arthritic joints, Bis-5-HT can respond to the local MPO/hydrogen peroxide and induce the aggregation of PD5NPs or binding to neighboring extracellular matrix protein via radial formation, resulting in in-situ size amplification and impaired transportation of drug formulation via lymphatic vessels. Furthermore, these aggregates can serve as drug depots for sustained Dex release under local acidic pH in arthritic joints, persistently mitigating RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn our study, the MPO responsive in situ size amplification behavior of PD5NPs was confirmed and the \u003cem\u003ein vivo\u003c/em\u003e performance of PD5NPs, encompassing pharmacokinetics, biodistribution, transportation and therapeutic efficacy were systematically investigated. More importantly, we extensively elucidated how the size amplification behavior affected the transportation of PD5NPs from inflamed joints to lymphatic vessels, and revealed the effectiveness of restraining lymphatic clearance for prolonged RA remission. To our knowledge, this in-situ size amplification strategy has never been applied in the treatment of inflammatory diseases. Our findings can not only offer an effective strategy for RA treatment, but also provide a new strategy to optimize the in vivo performance of nanomedicine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eFmoc-Glu-OH was purchased from Bide Pharmatech Ltd. (Shanghai, China). 5-hydroxytryptamine (5-HT) was purchased from Sigma-Aldrich (Chengdu, China). 1-ethyl-3-(3-dimethyl amino propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Macklin (Shanghai, China). Myeloperoxidase (MPO) was purchased from Sino Biological Inc. (Beijing, China). Dexamethasone (Dex) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were obtained from Aladdin Reagent, Ltd (Shanghai, China). Polysialic acid (PSA) was bought from Carbosynth China Ltd (Suzhou, China). Cy5.5-NH\u003csub\u003e2\u003c/sub\u003e was obtained from Meilun (Dalian, China). Complete Freund's adjuvant (CFA) was provided by Chondrex (Washington DC, USA). All reagents and solvents were purchased from Kemiou (Tianjin, China). Male SD rats were provided by Dashuo experimental animal center (Chengdu, China). All animal studies were approved by the Ethics committee of Sichuan Provincial People's Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of Bis-5HT-Glu-Fmoc:\u003c/h2\u003e \u003cp\u003eBriefly, N-hydroxysuccinimide (3 mmol) and EDC (3 mmol) were mixed with Fmoc-Glu-OH (1 mmol) in dimethylformamide (DMF, 30 mL) at room temperature and stirred for 30 min. Subsequently, 5-hydroxy tryptophan (5-HT, 2 mmol) and triethylamine dissolved in DMF (5 mL) were introduced into the above mixture. The reaction mixture was stirred for 2 h. The product was purified via reversed-phase chromatography and validated by \u003csup\u003e1\u003c/sup\u003eHNMR (400 MHz, Bruker AMX-400, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis of Bis-5HT-Glu-NH\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eBis-5HT-Glu-Fmoc (1 mmol) dissolved in 30 mL of DMF was mixed with piperidine (5 mmol) under stirring, and the mixture was stirred at room temperature for 2 h. DMF was removed under reduced pressure, and the residue was subsequently purified by reversed-phase chromatography to yield the compound Bis-5HT-Glu-NH\u003csub\u003e2\u003c/sub\u003e. The final product was obtained and confirmed by \u003csup\u003e1\u003c/sup\u003eHNMR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Synthesis of PSA-Dex-Bis-5HT\u003c/h2\u003e \u003cp\u003eInitially, Dex-NH\u003csub\u003e2\u003c/sub\u003e was synthesized according to previous established procedure [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Dex (5 mmol) and hydrazine hydrate (15 mmol) were dissolved in 30 mL of methanol, and a slow addition of 30 \u0026micro;L acetic acid preceded a reflux reaction at 80\u0026deg;C for 5 h. After the removal of unreacted substances through dialysis against ethanol and deionized water, Dex-NH\u003csub\u003e2\u003c/sub\u003e was obtained through lyophilization and subsequently dissolved in DMSO for subsequent reaction. In a parallel reaction, PSA (0.3 mmol disaccharide repeats), EDCI (1.5 mmol), and NHS (1.5 mmol) were dissolved in 40 mL of deionized water under stirring for 2 h to activate carboxyl groups. Subsequently, Bis-5HT-Glu-NH\u003csub\u003e2\u003c/sub\u003e (3 mmol) and Dex-NH\u003csub\u003e2\u003c/sub\u003e (3 mmol) were added and continuously stirred for 24 h. The resulting mixture underwent purification via dialysis against deionized water. The final product was obtained through lyophilization, and the chemical structure of PSA-Dex-Bis-5HT was confirmed by \u003csup\u003e1\u003c/sup\u003eHNMR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Preparation and characterization of PD5NPs\u003c/h2\u003e \u003cp\u003eIn brief, PSA-Dex-Bis-5HT was dissolved in methanol, and a dry thin film was formed through rotary evaporation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Subsequently, the thin film was rehydration with deionized water to produce a PD5NPs dispersion. The particle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS) with a Zetasizer Nano instrument (Anton Paar, Austria). The morphology of PD5NPs was visualized through transmission electron microscopy (TEM, JEOL TEM-1011, Japan). The conjugation ratio of Dex in PSA-Dex-Bis-5HT was assessed by high-performance liquid chromatography (HPLC, Agilent 1260, USA) following hydrochloric acid (HCl, 0.1 M) treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Stability evaluation\u003c/h2\u003e \u003cp\u003eTo assess the in vitro stability of PD5NPs, dynamic light scattering (DLS) was employed to measure particle size and polydispersity index (PDI) over time. The stability in serum was investigated through monitoring the change in particle size and PDI of PD5NPs incubated with rat serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In situ size amplification and characterization of aggregates\u003c/h2\u003e \u003cp\u003ePD5NPs solution were incubated with PBS containing MPO (at concentrations of 10 U) and 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. The PSA-Dex nanoparticles (PDNPs) without Bis-5HT modified was selected as control group. The alterations in size distribution and morphology were monitored using both DLS and TEM. The zeta potential of aggregates was determined by DLS. To evaluate the stability in serum of prepared aggregates, TEM was employed to measure the particle size and morphology over time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. In vitro release assay\u003c/h2\u003e \u003cp\u003eTo assess the pH-dependent release of Dex, PD5NPs and prepared aggregates were resuspended in PBS with different pH values. Subsequently, each sample was introduced into a dialysis bag and placed in a glass bottle containing release medium with diverse pH conditions. The in vitro release assay was carried out under gentle agitation at 37\u0026deg;C. At specified time intervals, aliquots of the release medium were collected, and the Dex concentration was quantified using HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Cell cytotoxicity\u003c/h2\u003e \u003cp\u003eTo determine the cytotoxicity of PD5NPs on human umbilical vein endothelial cells (HUVECs) and Raw264.7, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cell per well were seeded in 96-well plates were treated with 100 \u0026micro;L of PD5NPs and prepared aggregates at final concentrations ranging from 7.81 to 500 \u0026micro;g/mL. After 24 h of incubation, MTT solution (20 \u0026micro;L, 5 mg/mL) was added and incubated for 4 h. DMSO was then added to dissolve the blue formazan crystals. The absorbance of each well was measured using a microplate reader at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Establishment of arthritis model\u003c/h2\u003e \u003cp\u003eThe Adjuvant-Induced Arthritis (AIA) model was induced in male Sprague-Dawley rats (8\u0026ndash;10 weeks) using Complete Freund's Adjuvant (CFA) containing 10 mg/mL of mycobacterium tuberculosis. Specifically, 50 \u0026micro;L of CFA was subcutaneously injected into the hind paw of each Sprague-Dawley rat. The swelling of all rat limbs was monitored every other day post-induction. All animals were maintained under standard conditions with ad libitum access to food and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. MPO detection in AIA rats\u003c/h2\u003e \u003cp\u003eTo compare the MPO level between healthy and arthritic rats, MPO concentration in arthritic joints and healthy joints were determined using an ELISA kit. Arthritic rats exhibited observable joint redness and swelling were sacrificed and ankle joint tissues were dissected. The MPO concentration in joint homogenate was measured according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. In vivo biodistribution study\u003c/h2\u003e \u003cp\u003eTo elucidate the in vivo biodistribution of PD5NPs, Cy5.5-labeled formulations were employed for visualized observation using an in vivo imaging system (IVIS, Perkin Elmer, USA). The Cy5.5-labeled PDNPs were chosen as the control group. AIA rats were randomly assigned to three groups and intravenously injected with free Cy5.5, Cy5.5-PDNPs, or Cy5.5-PD5NPs. At specified time points, fluorescence signal in inflamed joints in AIA rats, as well as the fluorescence distribution in major organs and joint tissues were measured by IVIS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Co-localization of blood vessel\u003c/h2\u003e \u003cp\u003eTo investigate the colocalization of PD5NPs with hyperplastic vessels in inflamed synovium, rats receiving Cy5.5-PDNPs or Cy5.5-PD5NPs were euthanized at indicated time points, and synovium tissues were collected for preparing frozen sections. The obtained cryosections of synovium samples were fixed by 4% paraformaldehyde for 10 min and blocked with 5% fetal bovine serum. Subsequently, anti-CD31 antibody (Abcam, USA) was incubated with slices at 4\u0026deg;C overnight, followed by IgG-AlexaFluor 488 (Abcam, USA) incubation for 30 min. DAPI was used for nucleus staining. The colocalization of Cy5.5-PD5NPs with CD31-labeled vessels was then observed using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Pharmacokinetic behavior of Dex in plasma and inflamed joints\u003c/h2\u003e \u003cp\u003eAIA rats were randomly allocated into three groups and intravenously administrated with one of the following: free Dex, PDNPs, or PD5NPs at a Dex dosage of 2 mg/kg. At predetermined intervals, rats from each group were euthanized to collect both blood samples and joint tissues. The concentration of Dex in plasma and joint homogenates was quantified using liquid chromatography mass spectrometry (LC-MS/MS, Agilent, USA). Pharmacokinetic parameters, including the area under the concentration-time curve (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e), half-life (T\u003csub\u003e1/2\u003c/sub\u003e), and mean retention time (MRT\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e) were analyzed using DAS software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Therapeutic efficacy\u003c/h2\u003e \u003cp\u003eAIA rats were randomly assigned to four groups, with healthy rats serving as the normal control (n\u0026thinsp;=\u0026thinsp;5). AIA rats received intravenous injection of one of the following: PBS, free Dex, PDNPs, and PD5NPs (at a Dex dose of 2 mg/kg) every other day for three times. During the period of the treatments, the body weight, paw thickness measured via vernier caliper and joint score of the AIA rats were monitored every day in each group. The joint score determined by grading from score of 0 (no observable erythema or swelling) to 4 (severe swelling and erythema) was given for each paw. The swelling and ulceration degree of 4 paws were scored, finally resulting in a maximum possible score of 16 for each animal [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. After a two-week period, rats were euthanized and joints tissues were collected for subsequent pathological examination. The joint tissues from each group were cut up and homogenized. The concentration of inflammatory cytokines (TNF-α and IL-1β) in joint homogenates was measured by ELISA kits. The joint ankles from each group were soaked in 4% paraformaldehyde solution for three days and then incubated in 15% EDTA-2Na decalcification solution until the samples could be easily sliced. The joint slices were finally stained with hematoxylin and eosin to observe the inflammatory cell infiltration and cartilage erosion. For toluidine blue staining and safranin solid green staining, slices were incubated with specific reagent and observed via microscopy to analyze the cartilage integrity and tissue damage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. The colocalization of lymphatic vessels and PD5NPs\u003c/h2\u003e \u003cp\u003eAIA rats were randomly allocated into three groups and were administrated intravenously one of the following: free Cy5.5, Cy5-PDNPs, or Cy5.5-PD5NPs. At predetermined time points, rats were euthanized, and synovium tissues were collected. Cryosections of synovium tissues were sequentially incubated with anti-LYVE-1 antibody (Abcam, USA) and IgG-Alexa Fluor 488 for specific labeling of lymphatic vessels. DAPI was added for nucleus staining. All samples were examined using a fluorescence microscope. Colocalization analysis was conducted using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Synovium interstitial lymphatic inhibition\u003c/h2\u003e \u003cp\u003eInterstitial lymphatic growth in inflamed synovium was inhibited using the small molecule MAZ51. MAZ51 dissolved in DMSO (100 \u0026micro;g/mL) was loaded into a 29-gauge insulin syringe and intra-articular administered to joints. An intra-articular injection of the MAZ51(5 \u0026micro;L) ensures all drug molecules are within the target site while minimizing off-target effects. Meanwhile, AIA rats with intra-articular injection of PBS were selected as control. This intra-articular injection of MAZ51 was repeated daily for five days. PDNPs and PD5NPs were intra-articular administered on the sixth day. Ankle joint with the surrounding tissues and lymph nodes (popliteal and inguinal lymph nodes) were resected after 4 h and homogenized for LC-MS/MS detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18. In vivo safety evaluation\u003c/h2\u003e \u003cp\u003eThe body weight of each rat was monitored throughout the treatment period. Rats were euthanized to collect blood samples for blood routine test and biochemical analysis, encompassing assessments of alanine transaminase (ALT), aspartate aminotransferase (AST), as well as blood urea nitrogen (BUN) and creatinine (Cr) levels. Additionally, major organs were harvested and subjected to hematoxylin and eosin (H\u0026amp;E) staining for histopathological examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of minimum three replicates as indicated. Comparisons among multiple groups were expressed by one-way ANOVA. The significant difference between two comparative groups was analyzed by student\u0026rsquo;s t-test. All statistical analysis were performed using Graphpad Prism 7.0, P value of \u0026lt;\u0026thinsp;0.05 was considered as significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Synthesis and characterization of PD5NPs and aggregates\u003c/h2\u003e \u003cp\u003eThe synthesis of PSA-Dex-Bis-5HT involved a three-step process as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-2. Successful synthesis of Bis-5HT-Glu-NH\u003csub\u003e2\u003c/sub\u003e was evidenced by the disappearance of characteristic peaks at 4.29\u0026ndash;4.2 ppm (m, 2H) and 4.01\u0026ndash;3.93 ppm (m, 1H) in the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum (Figures S3). The \u003csup\u003e1\u003c/sup\u003eHNMR spectrum of PSA-Dex-Bis-5HT exhibited distinctive signals at 10.47 ppm (s, 2H), 8.02\u0026ndash;7.98 ppm (m, 1H), and 7.34\u0026ndash;7.44 ppm (m, 1H), which were absent in the spectrum of PSA alone (Figures S4). These findings demonstrated the successful synthesis of PSA-Dex-Bis-5HT.\u003c/p\u003e \u003cp\u003eIt was hypothesized that in the presence of MPO and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), Bis-5HT on the surface of PD5NPs can undergo oxidation and radical generation, which can subsequently engage in particle oligomerization and binding to phenolic residues from the proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Firstly, PD5NPs were prepared using a thin film hydration method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The average size and PDI of prepared PD5NPs exhibited minimal changes even after one week, indicating robust stability under static condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). When exposed to plasma condition at room temperature, no significant change in particle size or PDI were observed throughout the observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting favorable stability of PD5NPs in serum. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, PD5NPs exhibited a particle size approximately 150 nm with a narrow distribution, as determined by dynamic light scattering (DLS) at the initial time. After MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 4 h, the particle size of PD5NPs significantly increased and the size distribution become wide and disordered (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In accordance with DLS measurement, TEM images of PD5NPs revealed a uniform and near-spherical morphology in the absence of MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and Figure S5, obvious and widespread particle aggregation could be seen in different fields. Moreover, the zeta potential of the aggregates was about \u0026minus;\u0026thinsp;22.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mV measured using DLS. When incubated in serum, no significant morphology change of aggregates was observed throughout the observation period according to TEM results, suggesting favorable stability of these aggregates in serum (Figure S6). TEM images showed that the size of aggregates in serum was approximate range from 500 nm to 3 \u0026micro;m. In our study, PDNPs without Bis-5HT modification was used as a control. PDNPs showed similar particle size as PD5NPs (Figure S7). When incubated with MPO and hydrogen peroxide for 4 h and 24 h, the particle size of PDNPs remained no significant change (Figure S8). In contrast, the size distribution of PD5NPs underwent a marked increase after incubation with MPO and hydrogen peroxide for 4 h via DLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). These results indicated that Bis-5HT is necessary for aggregation.\u003c/p\u003e \u003cp\u003eThe Dex conjugation ratio to PD5NPs was calculated to be approximately 7.5%, as assessed by HPLC. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and Figure S9, in vitro release studies revealed that both PD5NPs and aggregates displayed a slow and sustained release profile in PBS at pH 7.4, whereas Dex release was notably accelerated under acidic pH condition. Furthermore, both Raw264.7 and HUVEC cell lines exhibited favorable cell viability when incubated with PD5NPs and aggregates over a concentration range of 7.81 to 500.00 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J and Figure S10). Hence, it can be concluded that PD5NPs and aggregates demonstrate excellent cytocompatibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Inflammation targeting behavior of PD5NPs\u003c/h2\u003e \u003cp\u003eTo assess the targeting ability of PD5NPs to arthritic joints, Cy5.5-PD5NPs were administered intravenously to adjuvant-induced arthritis (AIA) rats, and the biodistribution of PD5NPs was monitored using an in vivo imaging system (IVIS). The Cy5.5-labled PSA-Dex nanoparticles (PDNPs) without 5-HT modification were chosen as the control group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Figure S11, the fluorescent signal in the inflamed joints of rats receiving free Cy5.5 was barely detectable, with predominant fluorescence observed in the liver and kidney. In contrast, intensive fluorescent signal was observed at arthritic sites in rats treated with Cy5.5-PD5NPs and Cy5.5-PDNPs at 2 h post-injection. Nevertheless, fluorescent signal in rats receiving Cy5.5-PD5NPs remained remarkable even after 24 h, while rats receiving Cy5.5-PDNPs displayed very limited signal. The fluorescent quantitative analysis provided more direct comparison. The fluorescent intensity of joints in Cy5.5-PD5NPs group is nearly 2-fold higher than that in PDNPs group at 24 h post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This considerable difference in fluorescent intensity at 24 h between Cy5.5-PD5NPs and Cy5.5-PDNPs might be due to the delayed lymphatic transportation endowed by in-situ size amplification of PD5NPs. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC further corroborated this assumption. The arthritic joints from AIA rats exhibited a substantial increase in MPO expression compared to that from normal rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, the more detailed colocalization of Cy5.5-PD5NPs and blood vessel within inflamed tissues was visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. Consistent with IVIS findings, both PDNPs and PD5NPs treated groups displayed more robust fluorescence compared to free Cy5.5 group during our observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). At 6 h and 24 h, a significant overlap between red and green fluorescence indicated that both PDNPs and PD5NPs reached inflamed synovium through local hyperplastic vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). Both the Pearson\u0026rsquo;s coefficient for Cy5.5-PDNPs and Cy5.5-PD5NPs remained around 0.8 at 6 and 24 h, indicating a high degree of overlap with hyperplastic vessels (Figure S12). However, at 24 h, the colocalization areas of red and green fluorescence diminished, suggesting drug formulations gradually exited from inflamed synovium. Notably, the PD5NPs treated group exhibited much stronger red fluorescence than the PDNPs group at 24 hours after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We supposed that the in-situ size amplification of PD5NPs in response to MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can significantly enhance drug retention in arthritic sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.3. In vivo pharmacokinetics behavior of PD5NPs\u003c/h2\u003e \u003cp\u003eTo further investigate the \u003cem\u003ein vivo\u003c/em\u003e behaviors including pharmacokinetic and synovium retention of PD5NPs in arthritic joints, more accurate in vivo quantitative analysis was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The plasma pharmacokinetic curve was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The calculated area under the curve (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e) and half-time (T\u003csub\u003e1/2\u003c/sub\u003e) of PDNPs and PD5NPs significantly increased compared to free Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D), while the difference in MRT\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e between PDNPs and PD5NPs was not obvious in plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results suggested they can similarly enhance the Dex bioavailability and extend the \u003cem\u003ein vivo\u003c/em\u003e circulation time. However, a notable contrast in pharmacokinetic behavior emerged in inflamed joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Despite rapid decline in Dex concentration over time in both groups, PD5NPs exhibited a Dex concentration of nearly 12 ng/mL within inflamed joints after 36 h post administration, while only 2 ng/mL was observed in PDNPs groups. Furthermore, PD5NPs demonstrated a significant increase in AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e in joints compared to the PDNPs groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). The T\u003csub\u003e1/2\u003c/sub\u003e of Dex in inflamed joints in PD5NPs treated group was nearly up to 10 h, which was much higher than that in the PDNPs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The MRT\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e of the PD5NPs group was almost 2-fold higher than that in PDNPs treated group, indicating the significantly prolonged drug retention within arthritic joints after PD5NPs injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). These might be due to the in-situ size amplification of PD5NPs from small nanoparticles to larger aggregates in response to MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.4. In vivo lymphatic transport\u003c/h2\u003e \u003cp\u003eNanoparticles typically exhibit directional movement from areas of higher pressure to those of lower pressure. Microvascular pressure falls within the range of 2\u0026ndash;40 mmHg [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], while the internal pressure of lymphatic vessels is quite lower, generally about \u0026minus;\u0026thinsp;2 mmHg [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This creates a pressure gradient that predominantly guides the movement of nanoparticles toward lymphatic capillaries. Additionally, lymphatic vessels are distinguished from blood vessels due to the presence of specialized ultrastructural attributes including channel networks and a series of interconnected vesicles [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], which are responsible for trans-endothelial transport of macromolecule. Therefore, we can infer that PDNPs and PD5NPs might enter inflamed sites through blood capillaries but are probably transported out of synovium via lymphatic capillaries. To figure out whether PD5NPs can delay the lymphatic clearance, we explored the colocalization of lymphatic vessels with Cy5.5-PD5NPs after injecting Cy5.5-PD5NPs to arthritic rats as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB illustrates a rapid decrease in free Cy5.5 signal, with fluorescence becoming undetectable within 24 h. Both Cy5.5-PDNPs and Cy5.5-PD5NPs significantly prolonged drug retention within inflamed joints, with PD5NPs exhibiting a substantially higher fluorescence intensity than PDNPs at the 24 h (Figure S13). The colocalization degree of lymphatic vessels and Cy5.5-PDNPs was notably more pronounced than that of Cy5.5-PD5NPs, suggesting higher distribution of Cy5.5-PDNPs within lymphatic tissues. Additional colocalization analysis of red and green fluorescence at 24 h further revealed that PD5NPs had less colocalization with lymphatic vessels than PDNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Pearson\u0026rsquo;s coefficient, which reflects the degree of colocalization between the red and green signals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], also confirmed this observation. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, the Pearson\u0026rsquo;s coefficient for Cy5.5-PDNPs remained around 0.8 at both 6 and 24 h, indicating a high degree of overlap with lymphatic vessels. Conversely, the Pearson\u0026rsquo;s coefficient for Cy5.5-PD5NPs was approximately 0.2 at 24 h, denoting minimal distribution in lymphatic vessel. These results collectively verified that PD5NPs were able to restrain the lymphatic transport and delay the lymphatic clearance compared with PDNPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn general, nanoparticles in inflamed sites can be transported to lymphatic system via paracellular transport and transcellular transport [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The pressure gradient between microvasculature and lymphatic vessels, and the existence of lymphatic endothelial gap, usually contribute to the paracellular transport of nanoscale particles from interstitial space to lymph nodes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Vascular endothelial growth factor receptor 3 (VEGFR3) is predominantly expressed on lymphatic endothelial cells and plays a key role in lymph angiogenesis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The small molecule MAZ51 has been demonstrated to effectively disrupt lymph angiogenesis-driven pathology by antagonizing VEGFR-3 signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], thereby interfering the lymph angiogenesis in synovium. The proposed transport pathway of PDNPs and PD5NPs via lymphatic vessels were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. To further elucidate the transport ability of PD5NPs to lymph vessels in inflamed joints, we conducted a comparative analysis of drug transport from synovium to lymphatic systems by administering the joint with and without the VEGFR3-specific inhibitor MAZ51 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, the MAZ51 treatment resulted in significantly elevated PDNPs retention in inflamed joints and decreased accumulation in lymph nodes, suggesting that PDNPs might be mainly cleared from synovium through angiogenesis of lymph vessels. Moreover, MAZ51 treatment had no impact on the PD5NPs retention in inflamed joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), further implying that the transport process of PD5NPs from inflamed synovium to lymphatic vessels was almost inhibited even in the absence of MAZ51. Compared with PBS treatment, MAZ51 can inhibit lymph angiogenesis and slow down the rate of lymphatic vessels-dependent transport. The lymphatic transport of large-sized aggregates is relatively reduced compared with smaller particles. And the MAZ51-mediated shrinkage of lymphatic vessels might further interfere the transportation of PD5NPs aggregates towards lymphatic system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Collectively, based on these findings, we can speculate that the clearance of PDNPs mainly depends on the lymphatic transport, while PD5NPs appeared to greatly inhibit the lymphatic clearance via in-situ size amplification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Therapeutic efficacy\u003c/h2\u003e \u003cp\u003eThe therapeutic efficacy of PD5NPs was investigated in an AIA rat model and the treatment regimen was outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA. The AIA model was successfully established and AIA rats were randomly allocated into four groups and administered PBS, free Dex, PDNPs, or PD5NPs at a Dex dosage of 2 mg/kg on days 8, 10, and 12, respectively. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the hind paws of rats in PBS treated group exhibited pronounced swelling and ulceration. Treatment with PDNPs resulted in a modest reduction in swelling, whereas PD5NPs treatment significantly mitigated joint swelling, restoring the joints to nearly normal condition. The progression of joint scores and paw thickness among all groups corresponded closely with the visual assessment of joint morphology, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D. Compared to PBS group, free Dex treatment achieved a marginal decrease in joint score and paw thickness. In contrast, PD5NPs effectively abrogated joint swelling and symptoms of RA. The area under the curve (AUC) analysis for joint score (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and paw thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) showed a direct and quantitative comparison among these treatment groups, corroborating the abovementioned observation. Furthermore, the levels of pro-inflammatory cytokines in arthritic joints, tumor necrosis factor-alpha (TNF-α) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), and interleukin-1 beta (IL-1β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH), were significantly elevated in PBS treated group of AIA rats. Conversely, PD5NPs treatment exhibited the lowest levels of TNF-α and IL-1β among all groups, underscoring the potent anti-inflammatory property of PD5NPs.\u003c/p\u003e \u003cp\u003eTo elucidate the histopathological changes after various therapeutic interventions, the ankle joints from all experimental rats were sectioned and subjected to microscopic examination. Hematoxylin and eosin (H\u0026amp;E) staining of the articular tissues from PBS treated group disclosed a marked constriction of the joint space, accompanied by pervasive inflammatory cell infiltration and pronounced cartilage degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). Conversely, PD5NPs treated group exhibited a remarkable restoration of the articular space, negligible inflammatory infiltration, and only minimal sign of cartilage damage. For a more detailed assessment of cartilage integrity and tissue damage within the joint tissues, toluidine blue staining was utilized [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI (middle) revealed significant cartilage deterioration and fibrotic overgrowth in PBS treated group, indicating serious articular damage. In contrast, PD5NPs treatment markedly attenuated cartilage erosion. Additionally, the integrity of the cartilaginous matrix was evaluated using safranine O and fast green (SO\u0026amp;FG) staining [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. PBS group displayed a paucity of red staining, reflecting substantial cartilage depletion and osseous tissue necrosis. In comparison, the PD5NPs group manifested an extensive red staining area, denoting robust cartilage preservation and potential reparative activity. Collectively, these results demonstrated that PD5NPs not only ameliorate synovial inflammation but also significantly inhibit cartilage degradation in the AIA rat model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.6. In vivo biocompatibility evaluation\u003c/h2\u003e \u003cp\u003eTo further elucidate the in vivo biocompatibility of PD5NPs, rats were sacrificed for a safety evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Throughout the treatment regimen, rats in all groups exhibited continuous body weight gain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Furthermore, there was no difference among all groups in terms of red blood cell (RBC), white blood cell (WBC) and platelet (PLT) level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-E), suggesting good compatibility of our treatment. Likewise, there was no significant variance observed in the serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF-I), indicating that our treatment did not induce liver or renal damage. Additionally, the H\u0026amp;E staining showed that no inflammation infiltration or cellular necrosis was detected in major organs among all groups (Figure S14). Hence, our treatment demonstrated good biocompatibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, we developed an in-situ size amplification strategy with the objective of suppressing in vivo lymphatic clearance and extending the therapeutic efficacy in RA treatment. The prepared PD5NPs can transform from small-sized nanoparticles to larger-sized aggregates in the presence of MPO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in inflamed sites. Following intravenous administration, PD5NPs exhibited prolonged blood circulation in vivo and increased retention at inflamed joints. Notably, PD5NPs effectively impeded drug clearance through lymphatic system by undergoing in-situ size amplification into aggregates in response to inflammatory condition. In the arthritic model, PD5NPs efficiently attenuated RA development and promoted cartilage tissue repairing. Altogether, this in situ size amplification strategy exhibited great potential to enhance the therapeutic efficacy in RA treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were approved by the Ethics committee of Sichuan Provincial People's Hospital before starting any animal experiments. All experiments were conducted following the guidelines of the National Institute of Health for the ethical care and handling of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors concur with the submission and publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supporting information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting data to this article can be found online at xxx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by\u0026nbsp;the\u0026nbsp;National Natural Science Foundation of China (No. 82404541, No. 82003661 and No.82271120), Postdoctoral Fellowship Program of CPSF under Grant Number GZC20240218, Postdoctoral Fund of Sichuan Provincial People's Hospital (No.2023BH10), Natural Science Foundation of Sichuan Province of China (No. 2023NSFSC1679), Sichuan Science and Technology Program (No. 2022ZYD0131), the CAMS Innovation Fund for Medical Sciences (No.2019-I2M-5-032), and the Department of Science and Technology of Sichuan Province, China (No.2024ZHYS0018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYi Shi\u0026nbsp;and\u0026nbsp;Qin Wang\u0026nbsp;conceived and supervised the research.\u0026nbsp;Xianyan Qin and Luhan Zhang\u0026nbsp;carried out the experiments and performed data analysis.\u0026nbsp;Yang-Bao Miao and Linxi Jiang\u0026nbsp;participated in part of the experiments.\u0026nbsp;Liang Zou\u0026nbsp;provided intellectual discussions on experimental designs.\u0026nbsp;Xianyan Qin\u0026nbsp;wrote the manuscript.\u0026nbsp;Qin Wang,\u0026nbsp;Yang-Bao Miao\u0026nbsp;and\u0026nbsp;Yi Shi\u0026nbsp;revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDeane KD, Holers VM, Rheumatoid Arthritis Pathogenesis, Prediction, and Prevention: An Emerging Paradigm Shift. \u003cem\u003eArthritis Rheumatol.\u003c/em\u003e 2021;73:181-93.\u003c/li\u003e\n\u003cli\u003eKomatsu N, Takayanagi H, Mechanisms of joint destruction in rheumatoid arthritis - immune cell-fibroblast-bone interactions. \u003cem\u003eNat. Rev. Rheumatol.\u003c/em\u003e 2022; 18:415-29.\u003c/li\u003e\n\u003cli\u003eFang RH, Zhang L, Biohybrid nanoparticles for treating arthritis. \u003cem\u003eNat Nanotechnol \u003c/em\u003e 2023; 18:1387-8. \u003c/li\u003e\n\u003cli\u003eNooreen R, Nene S, Jain H, Prasannanjaneyulu V, Chitlangya P, Otavi S, Khatri DK, Raghuvanshi RS, Singh SB, Srivastava S, Polymer nanotherapeutics: A versatile platform for effective rheumatoid arthritis therapy. \u003cem\u003eJ. Control. 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Commun.\u003c/em\u003e 2018; 9:1296-302.\u003c/li\u003e\n\u003cli\u003eYamamura A, Nayeem MJ, Muramatsu H, Nakamura K, Sato M, MAZ51 Blocks the Tumor Growth of Prostate Cancer by Inhibiting Vascular Endothelial Growth Factor Receptor 3. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e 2021; 20:667474-86.\u003c/li\u003e\n\u003cli\u003eSchmitz N, Laverty S, Kraus VB, Aigner T, Basic methods in histopathology of joint tissues. \u003cem\u003eOsteoarthr. Cartilage \u003c/em\u003e 2010; 18:113-6.\u003c/li\u003e\n\u003cli\u003eRutgers M, Pelt MJP, Dhert WJA, Creemers LB, Saris DBF, Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage. \u003cem\u003eOsteoarthr. Cartilage\u003c/em\u003e 2010; 18:12-23.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rheumatoid arthritis, nanomedicine delivery, in situ size amplification, aggregates, lymphatic clearance","lastPublishedDoi":"10.21203/rs.3.rs-5069556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5069556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRheumatoid arthritis (RA) is an autoimmune condition causing painful swelling and inflammation due to immune system attacks on healthy cells. However, arthritic sites often experience increased lymph flow, hastening drug clearance and potentially reducing treatment effectiveness. To address this challenge, an in situ size amplification has been proposed to inhibit lymphatic clearance and thereby enhance arthritis therapy. This system has been developed based on a conjugate of dexamethasone (Dex) and polysialic acid (PSA), linked via an acid-sensitive linker, supplemented with bis-5-hydroxytryptamine (Bis-5HT) on the PSA backbone. Under physiological conditions, the system autonomously assembles into stable nanoparticles (PD5NPs), facilitating prolonged circulation and targeted delivery to inflamed joints. Upon arrival at arthritic joints, Bis-5HT reacts to elevated myeloperoxidase (MPO) levels and oxidative stress, prompting particle aggregation and in-situ size amplification. This in situ size amplification nanocarrier effectively inhibits lymphatic clearance and serves as reservoirs for sustained Dex release in acidic pH environments within arthritic sites, thus continuously alleviating RA symptoms. Moreover, investigation on the underlying mechanism elucidates how the in situ size amplification nanocarrier influences the transportation of PD5NPs from inflamed joints to lymphatic vessels. Our study offers valuable insights for optimizing nanomedicine performance in vivo and augmenting therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"In Situ Size Amplification Strategy Suppresses Lymphatic Clearance for Enhanced Arthritis Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 04:25:41","doi":"10.21203/rs.3.rs-5069556/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-22T02:46:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-09T23:15:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-09T08:00:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-08T05:06:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47343258837201710612333108059717346446","date":"2024-09-20T03:44:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329594284821174162910516847196775183086","date":"2024-09-19T14:58:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80748449228646010486521926994121555307","date":"2024-09-17T21:41:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170525532879577178128320711924547678496","date":"2024-09-17T19:14:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-17T14:03:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-13T17:18:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-13T17:18:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-09-11T08:43:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1dcd8aa1-6efb-418b-b8a2-472fddd5a774","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:01:23+00:00","versionOfRecord":{"articleIdentity":"rs-5069556","link":"https://doi.org/10.1186/s12951-024-03061-8","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-12-18 15:57:27","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-11-05 04:25:41","video":"","vorDoi":"10.1186/s12951-024-03061-8","vorDoiUrl":"https://doi.org/10.1186/s12951-024-03061-8","workflowStages":[]},"version":"v1","identity":"rs-5069556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5069556","identity":"rs-5069556","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}