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Secreted Protein Acidic and Rich in Cysteine Mediated Biomimetic Delivery of Methotrexate by AlbuminBased Nanomedicines for Rheumatoid Arthritis Therapy Lu Liu, Fanlei Hu, Hui Wang, Xiaoli Wu, Ahmed Shaker Eltahan, Stephanie Stanford, Nunzio Bottini, Haihua Xiao, Massimo Bottini, Weisheng Guo, and Xing-Jie Liang ACS Nano, Just Accepted Manuscript • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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Secreted Protein Acidic and Rich in Cysteine Mediated Biomimetic Delivery of Methotrexate by Albumin-Based Nanomedicines for Rheumatoid Arthritis Therapy Lu Liu,1.2.4 Fanlei Hu,5 Hui Wang,6 Xiaoli Wu,7 Ahmed Shaker Eltahan,3 Stephanie Stanford,8 Nunzio Bottini,8 Haihua Xiao,9 Massimo Bottini,1,4,* Weisheng Guo,3,* Xing-Jie Liang 1,2,* Affiliations: 1

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, P. R. China.

2

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

3

Translational Medicine Center, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510260, P. R. China.

4

Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, 00133, Italy.

5

Department of Rheumatology and Immunology, Peking University People's Hospital, Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis, Beijing, 100044, P. R. China

6

CAS Key Laboratory for Nanosystem and Hierarchy Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing 100190, P.R. China.

7

School of Life Sciences, Tianjin University, Tianjin 300072, P. R. China

8

Altman Clinical & Translational research Institute, University of California San Diego, La Jolla, CA 92037, USA

9

Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China

* To whom correspondence should be addressed: Weisheng Guo: [email protected]; XingJie Liang: [email protected]; Massimo Bottini: [email protected];

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ABSTRACT Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases. Despite considerable advances in clinical treatment of RA, suboptimal response to therapy and treatment discontinuation are still unresolved challenges due to systemic toxicity. It is of crucial importance to actively target and deliver therapeutic agents to inflamed joints in order to promote in situ activity and decrease systemic toxicity. In this study, we found that SPARC (secreted protein acidic and rich in cysteine) was overexpressed in the synovial fluid and synovium of RA patients as well as mice with collagen induced arthritis (CIA), which has been scarcely reported. Building upon the SPARC signature of RA joint microenvironment and the intrinsic high affinity of SPARC for albumin, we fabricated methotrexate-loaded human serum albumin nanomedicines (MTX@HSA NMs) and explored them as biomimetic drug delivery systems for RA therapy. Upon intravenous injection of chlorin e6-labeled MTX@HSA NMs into CIA mice, the fluorescence/magnetic resonance dual-modal imaging revealed higher accumulations and longer retention of MTX@HSA NMs in inflamed joints with respect to free MTX molecules. In vivo therapeutic evaluations suggested that the MTX@HSA NMs were able to attenuate the progression of RA with better efficacy and less side effects even at half dose of administrated MTX in comparison with free MTX. By unraveling the mechanism driving the efficient accumulation of MTX@HSA NMs in RA joints and showing their ability to improve the safety and therapeutic efficacy of MTX, our work sheds the light on the development of innovative anti-RA nanomedicines with a strong potential for clinical translation.

KEYWORD: Rheumatoid arthritis, SPARC, albumin, methotrexate, biomimetic delivery


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Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases, affecting 0.5% 1% of the world population with considerable morbidity. It is characterized by autoimmunity, severe synovial and systemic inflammation, bone erosion and cartilage damage.1-3 The current clinical approach for RA is focused on the alleviation of pain and minimization of joint damage.4,5 Methotrexate (MTX) is one of the most widely used first-line disease-modifying antirheumatic drugs (DMARD) in the clinical treatment of RA.6-8 MTX is a folic acid analogue that promotes an overall anti-inflammatory state even at low doses.9 Although the exact mechanism has not been completely elucidated, the adenosine signaling pathway is currently the leading hypothesis to explain the strong anti-RA effect of MTX.10 However, long-term systemic administration of MTX often results in suboptimal response and off-target systemic toxicities, including impaired immune system and opportunistic infections.4,11,12 Thus, it is of crucial importance to target and deliver therapeutic agents to arthritic joints with high efficiency and specificity in order to improve the therapeutic index and decrease systemic toxicities. Advances in nanotechnology, chemistry and biology have led to the emergence of nanomedicines (NMs).13-15 Cancer NMs display distinctive properties, including the ability of passive targeting and persisting in diseased sites by exploiting the leaky vasculature and poor lymphatic draining of tumor tissues, a phenomenon called as the enhanced permeability and retention (EPR) effect.16-18 The EPR effect has been recently exploited to passively target NMs to RA joints.15,19 Growing evidence has suggested that an EPR-like phenomenon is also a hallmark of RA, because the blood-joint barrier is disrupted as inflammation progresses, resulting in leaky blood vessels with inter-endothelial cell gaps up to 600 nm in inflamed joints.20,21 NMs also enable active targeting strategies when combined with the characteristic pathophysiological signatures of the diseased site microenvironment. The glycoprotein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin) is a member of the family of the matricellular components of the extracellular matrix (ECM).22 In normal tissues, SPARC is highly expressed in bones, teeth, adult eye, and at sites of wound repair and tissue


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remodeling where it modulates cell-ECM interactions.23 However, it has been well documented that SPARC is also overexpressed in atherosclerotic lesions and in the microenvironment of various aggressive cancers, such as melanomas, head & neck, breast, ovarian and colorectal cancer.24-29 Overexpression of SPARC is associated with increased tumor invasion and metastasis through its effects on matrix composition and cell adhesion.29,30 For example, SPARC can disrupt the integrity of vascular endothelial cell layers and thus facilitate the passage of melanoma cells and their metastases at distant sites.31,32 The discovery of the SPARC signature of the tumor microenvironment has contributed to the success of anti-cancer albumin-bound paclitaxel (Abraxane), which is the first nanotechnology-based drug on the market. The accumulation of Abraxane in the tumor microenvironment is facilitated by the overexpression of SPARC because of the high affinity of SPARC for albumin.33 Increased SPARC levels in tumors correlated with higher tumor accumulation and enhanced therapeutic efficacy of Abraxane.33,34 Despite the comprehensive investigations on SPARC-mediated anti-cancer therapies, little is known about the SPARC signature of the RA microenvironment, and indeed, the exploitation of the overexpression of SPARC in inflamed joints for efficient anti-RA therapeutic approaches has not yet been reported. Considering that SPARC was initially identified in bones and promotes tumor invasion,35,36 we hypothesized that SPARC levels in inflamed joints might increase with the invasion of inflammatory cells, neovascularization and bone erosion. We confirmed that SPARC is overexpressed in the synovial fluid and joint tissues from RA patients as well as mice with collagen induced arthritis (CIA). Building upon this finding and motivated by the intrinsic high affinity of SPARC for albumin,37,38 we fabricated methotrexate (MTX)-loaded human serum albumin (HSA) NMs (referred to as MTX@HSA NMs) for biomimetic drug delivery aimed at RA treatment (Scheme 1). In addition, because the metabolism of synovial cells is up-regulated, arthritic joints metabolize higher amounts of albumin than healthy tissues, using it as a nitrogen and energy source.39,40 The increased metabolism of albumin increases the demand for albumin by arthritic joints, which can further boost the biomimetic delivery of


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MTX@HSA NMs to these sites. Upon intravenous injection of MTX@HSA NMs into CIA mice, the fluorescence/magnetic resonance dual-modal imaging analysis revealed a superior accumulation and longer retention of MTX@HSA NMs in arthritic joints with respect to free HSA and MTX molecules. In vivo therapeutic evaluations revealed that the MTX@HSA NMs were able to attenuate the progression of RA with a higher therapeutic index and less side effects with respect to an equivalent dose of free MTX. Because of the clinical relevance of this work, we envision that the reported MTX@HSA NMs provide an innovative approach for the treatment of RA and have an attractive potential for translation.

RESULTS AND DISCUSSIONS Overexpression of SPARC in arthritic joints Since its initial identification in the bone, SPARC has been demonstrated to be overexpressed in various cancers, including melanoma, breast, prostate, esophagus, head & neck, liver and colorectal cancers.31,41 These pioneering studies suggested that SPARC is associated with increased tumor invasion, metastasis and poor prognosis.25,42 However, no evidence has yet been provided for an association of SPARC with RA. Considering the pathophysiological characteristics of RA, which include inflammation, cell invasion and bone erosion,1,3,43 we investigated SPARC expression in human synovial tissues by immunohistochemistry (IHC) and SPARC concentration in human synovial fluid by enzyme-linked immunosorbent assay (ELISA). Synovium samples from osteoarthritis (OA) patients were used as non-autoimmune controls, because samples from healthy humans were not available in the clinic. Higher SPARC expression was observed in the synovium of RA patients with respect to OA patients (Figure 1A). We also compared SPARC concentrations in the synovial fluid of 18 RA and 18 OA patients. As shown in Figure 1C, we found a significantly greater concentration of SPARC in RA individuals (224.8 ± 37.04 ng/mL) versus OA individuals (104.8 ± 23.37 ng/mL). In addition, the expression of SPARC was also investigated in healthy and collagen induced arthritis


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(CIA) mice (n = 3) by IHC and western blotting (WB). High SPARC immunoreactivity was detected in the synovium from CIA mice, while only a moderate expression of SPARC was found in the samples from healthy animals (Figure 1B). The WB data revealed about 2-fold greater SPARC expression levels in arthritic joints than in healthy ones (Figure 1D). The relative mRNA expression of SPARC in the arthritic ankles from CIA mice was also detected by RT-PCR tests with healthy joints as control. As presented in Figure S1 (in Supporting Information, SI), the inflamed ankles exhibited about 3-fold higher relative mRNA expression of SPARC than the healthy ones. Taken together, these results indicate that SPARC is overexpressed in joints with inflammatory arthritis.

Fabrication of MTX@HSA NMs for SPARC-mediated biomimetic drug delivery The overexpression of SPARC in arthritic joints encouraged us to use albumin-based NMs as biomimetic anti-RA drug delivery systems by exploiting the high affinity between SPARC and albumin.33 In addition, RA patients develop hypoalbuminemia presumably due to the increased albumin catabolism in arthritic joints.9 Because of the increased metabolism of synovial cells during RA, arthritic joints metabolize higher amounts of albumin than healthy tissues, using it as a nitrogen and energy source. The increased metabolism of albumin increases the demand for albumin by arthritic joints, thus further boosting the efficiency of biomimetic delivery of albumin-based NMs to these sites. Therefore, we prepared MTX@HSA NMs following a developed method.44,45 Transmission electron microscopy (TEM) images showed that the MTX@HSA NMs were spheroidal in shape and with a uniform size (30.71 ± 4.62 nm) (Figure 2A). Dynamic light scattering (DLS) analysis showed that the MTX@HSA NMs were well dispersed in PBS (1X, pH 7.4) with a hydrodynamic diameter (HD) of 116.20 ± 18.3 nm (Figure 2C). In order to better understand how the MTX@HSA NMs formed, the chemical interactions between HSA and MTX molecules were assessed by computer simulation. These studies showed that the interactions between HSA and MTX molecules were mainly hydrophobic force, thus we posited that these interactions were the driving force for the self-assembly of MTXloaded HSA into nanoparticles (Figure 2B, Figure S2 and Movie S1 in SI). We also assessed the


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secondary structure of HSA and MTX@HSA NMs by circular dichroism (CD). No significant differences were found in the CD spectra of HSA and MTX@HSA NMs, suggesting that the secondary structure of HSA was not affected by the formation of the nanoparticles (Figure 2D). The presence of MTX in the MTX@HSA NMs was validated by the characteristic absorption peak of MTX at 302 nm in the ultraviolet-visible (UV-Vis) absorbance spectrum of the nanoparticles (Figure 2E). The albumin concentration of the MTX@HSA NMs was determined by BCA kits. The extracted MTX from the MTX@HSA NMs was quantified via high performance liquid chromatography (HPLC) (as shown in Figure S3). The loading efficiency (LE) and loading capacity (LC) of MTX in MTX@HSA NMs was determined as 87.7 % and 4.55%, respectively. Finally, we evaluated the in vitro drug release behavior of MTX@HSA NMs in PBS at neutral (pH 7.4) and acidic (pH 5.8) pH. As shown in Figure 2F, MTX was released from the NMs at a higher rate in acidic conditions than in neutral conditions with no significant burst effect. This phenomenon was due to the changes in the conformation of HSA induced by the acidic pH. These changes altered the interactions between HSA and MTX and enabled the release of the drug from the NMs. Given the fabrication of MTX@HSA NMs, the interaction of HSA with SPARC was measured using microscale thermophoresis (MST). The estimated Kd was calculated to be approximately 44.79 ± 11.13 nM (Figure 2G), which confirmed the high affinity between HSA and SPARC.

In vivo SPARC-mediated biomimetic delivery of MTX@HSA NMs to arthritic joints Since Chlorine e6 (Ce6) is a near infrared (NIR) fluorophore as well as a good chelator for manganese (Mn) species, we used MTX@HSA NMs, free HSA and free MTX molecules covalently conjugated to Ce6 for the in vivo investigations. Thereby, the in vivo biodistribution behaviors of the compounds could be monitored by both fluorescence and magnetic resonance imaging (MRI) in a realtime and non-invasive manner. Similarly to MTX@HSA NMs, the Ce6-labelled MTX@HSA NMs had a spheroidal shape (Figure S4A) and an intense fluorescence peak centered at 680 nm (Figure


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S4B). The MTX@HSA NMs labelled with Ce6 and chelated with Mn showed a high R1 relaxivity of 13.83 mM-1·s-1 (Figure S4C, D), which suggested the potential use of these nanoparticles for MRI imaging. As depicted in Figure 3A, the CIA model was established by repeated immunization of DBA/1 male mice. Arthritis was fully developed two weeks after the second immunization, and mice were randomly divided into 4 groups (n = 3). Three groups were intravenously (i.v.) injected with 100 μL of either MTX@HSA NM, or free HSA or free MTX suspension, respectively, at an equal concentration of Ce6 (0.3 mg /mL). The mice were imaged at various time-points post-injection (p.i.) using an in vivo fluorescence imaging system (IVIS) to track the joint trafficking of the compounds. As shown in Figure 3B, the intensity of the fluorescence signal arising from the paws of free MTXinjected mice reached a maximum and decayed very rapidly after the injection. Compared with free MTX, the intensity of the fluorescence signal arising from the paws of HSA-injected mice showed a much lower decay rate (Figure 3D). The increased persistence time of HSA in the inflamed joints with respect to MTX was attributed to the high affinity between SPARC and HSA. Instead, the fluorescence signals arising from the paws of mice injected with MTX@HSA NMs increased during the initial 6 hours p.i. and remained fairly steady during the following 18 hours. Importantly, the paws of the mice injected with MTX@HSA NMs showed a significantly stronger fluorescence than the mice injected with free MTX (Figure 3D), which was due to the efficient exploitation of the EPR-like effect of arthritic joints by the nanoscale materials.20 For a comprehensive investigation of the effect of SPARC on the joint trafficking of MTX@HSA NMs, the binding between SPARC and HSA in the arthritic joints was blocked by incubating MTX@HSA NMs with excess of SPARC peptides for 1 hour in vitro in prior to injection into the fourth group of CIA mice pre-injected with an anti-SPARC antibody. When the binding between SPARC and HSA was blocked, the accumulation and retention of the NMs in the arthritic joints were significantly compromised (Figure 3D). It is worth noting that the accumulation of the NMs in arthritic joints was not completely abrogated by blocking the interaction between SPARC and HSA, since it was likely still favored by the increased demand for albumin by


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arthritic joints due to up-regulated metabolism of synovial cells. Therefore, the higher accumulation and longer retention of MTX@HSA NMs in the arthritic paws with respect to free MTX could be attributed to a combination of the EPR-like effect of arthritic joints,20 the SPARC-mediated biomimetic targeted delivery as well as the local increased demand for albumin.17,39,46 The main organs (heart, liver, spleen, lungs and kidneys) and paws of mice from the different groups were collected 24 hours p.i. and subjected to fluorescence analysis ex vivo. As displayed in Figure 3C and 3E, the paws from the mice injected with MTX@HSA NMs showed the highest fluorescence intensity. In parallel with the fluorescence detection, the accumulation efficiency of MTX in major organs and arthritic ankles was evaluated by percentage of injection dosage per gram (% ID/g) and measured by HPLC. The CIA mice injected with MTX@HSA NMs showed fairly good accumulation (0.86 ± 0.09 % ID/g) of MTX in inflamed ankles, while pretty low accumulations of free MTX in the arthritic ankle were beyond the MTX detection limit of HPLC (Figure S5). This result well agrees with the fluorescence imaging investigations in vivo and ex vivo. The pharmacokinetic profiles of MTX@HSA NMs and free MTX were also investigated by detecting the MTX concentration in plasma via HPLC. As revealed in Figure 3F, the MTX@HSA NMs displayed prominently extended blood circulation half-life (t1/2 ≈ 1.09 h) in respect to free MTX (t1/2 ≈ 7.0 min). The relatively longer circulation in blood contributed to the more efficient accumulation of MTX@HSA NMs at the arthritic joints. Finally, considering that MRI is one of the standard methods for RA diagnosis in the clinic, we imaged the arthritic paws before and after the systemic injection of MTX@HSA MNs labelled with Ce6 and chelated with Mn. As showed in Figure 3G and in the Movies S2 and S3, the inflammation in the ankle and phalangeal joints became more distinctive because of the enhanced contrast upon the administration of MTX@HSA NMs, making these nanoparticles a potential tool to provide accurate pathological information to doctors. To further assess the trafficking profile of the NMs in the synovium of arthritic joints, we systemically injected Ce6-labeled MTX@HSA NMs into both healthy and CIA mice and collected the ankles 4 hours after injection. Histological slides were prepared, co-stained for SPARC and imaged by


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immunofluorescence (IF) using a laser scanning confocal microscope (LSCM). In accordance with our previous results, the IF images revealed the presence of numerous SPARC proteins within the synovium of CIA mice (green dots in Figure 4A), but not in the ankles of healthy mice. Additionally, the IF images showed that the (red) fluorescence signal arising from the MTX@HSA NMs colocalized well with that arising from SPARC (green), suggestive of binding between the MTX@HSA NMs and SPARC. The colocalization of MTX@HSA NMs and macrophages in RA joints was also tested by immunofluorescence, as presented in Figure S6. The well colocalization between red signals (NMs) and green signals (macrophage) suggested the phagocytosis of NMs by macrophage. Hence, we posited that the NMs migrated into the arthritic joints through the leaky blood vessels attracted by the local increased demand for albumin and SPARC overexpression, and were taken up by activated synovial macrophages, which are the predominant cell population in arthritic joints.47 To further validate this model, MTX@HSA NMs were added to a culture of RAW 264.7 macrophage cells activated with lipopolysaccharide (LPS) for 24 hours. More efficient uptake of MTX@HSA NMs by activated macrophages with respect to non-activated cells was observed by LSCM and flow cytometry analyses after 4 hours of incubation (Figure S7). In addition, the MTX@HSA NMs appeared to enter activated macrophages through phagocytosis after 15 minutes of incubation, while much weaker fluorescence signal arising from MTX@HSA NMs was detected in non-activated macrophages (Figure 4B). Following the phagocytosis, the NMs were transported into the lysosomal compartments. We posited that the acidic microenvironment within the lysosomes could trigger the release of MTX from the NMs due to their pH-responsive drug release property (Figure 2F) and result in an efficient blockade of RA progression by inhibiting the proliferation of activated macrophages and expression of inflammatory cytokines (Figure S8).

In vivo therapeutic efficacy of MTX@HSA NMs Next, we investigated the therapeutic efficacy of the NMs on CIA mice following the treatment scheme outlined in Figure 5A. 28 days after the primary immunization, mice were randomly divided


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into 5 groups (referred to as G1 - G5, n=5 in each group), which were i.v. injected with PBS, free MTX solution (2.5 mg/kg or 5 mg/kg MTX per body weight) and MTX@HSA NM solution (2.5 mg/kg and 5 mg/kg of MTX per body weight), respectively. All the groups were treated every three days for six times (red arrows in Figure 5A). The arthritis severity was scored following a clinical scoring system (Figure S9) and the paw thickness was measured before the injections by means of a caliper. The therapeutic efficacy was characterized by the alleviation ratio (AR) calculated as the number of mice displaying a total clinical score smaller than 4 with respect to the total number of mice in the group. As shown in Figure 5B and 5C, the arthritis score of the paws rapidly increased in PBS-injected mice (group G1) with disease progression, while the increase in the arthritis score was much slower in mice injected with free MTX (groups G2 and G3) and MTX@HSA NMs (groups G4 and G5). Overall, the mice injected with MTX@HSA NMs (G4 and G5) exhibited significant lower clinical scores than the mice injected with an equivalent dose of free MTX (G2 and G3) at the end of the therapy (Figure 5B and 5D). In particular, more efficient progression inhibition was even realized by the treatment with MTX@HSA NMs at dose of 2.5 mg/kg (G4), which was only half of the administrated dose of MTX cocktail in G3 (G3 vs G4, *p = 0.0106). Additionally, the mice from group G4 exhibited an AR of ~ 4/5 compared to an AR of ~ 1/5 observed for the mice from group G3 (Figure 5B). Importantly, we observed severe alopecia in 3 mice from group G3, while no abnormal reactions were observed in mice from group G5 (Figure S10). This suggests that MTX@HSA NMs enabled safer treatment even at concentrations of MTX that led to obvious side-effects in free formulation. The changes in paw thickness during the treatment were also measured as an indicator of arthritis progression. As displayed in Figure 5D and 5E, the control mice (group G1) suffered from significant paw erythema and swelling. These signs were markedly relieved in the mice injected with MTX cocktails and MTX@HSA NMs. Of note, a more significant inhibition of the increase in paw thickness was observed on the mice who received MTX@HSA NMs treatment (G4) at half dose of MTX compared to MTX cocktail treatment (G3) (G3 vs G4, *p = 0.0302). To assess functional limitation of the mice, we kept


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each mouse in a separate chamber equipped with a running roller and an electronic monitor for 24 hours (Figure 5F and Movie S4). In accordance with the previous results, the mice treated with the MTX@HSA NMs showed a much stronger athletic ability than those treated with free MTX and PBS. Taken together, these lines of evaluations indicated that MTX@HSA NMs could achieve better alleviation efficacy and safer use even at half dose of administrated MTX in comparison with free MTX cocktail. The therapeutic efficacy of the treatments was further evaluated by MRI and micro computed tomography (CT) imaging. MRI can simultaneously assess the pathological functions and anatomical structure in the inflamed joints and has been widely used for clinical RA assessment post-treatments. CT is of importance for clinical RA diagnosis to assess bone erosion. As shown in Figure 6A, the inflammation levels of mice injected with MTX@HSA NMs (groups G4 and G5) was efficiently inhibited, whereas the control mice (group G1) showed obvious inflammation, as indicated by the strong T1 weight signals. The reconstructed CT images revealed that the ankle and finger joints of the control mice suffered serious bone erosion (Figure 6B). Reduced bone erosion activity was partially achieved by injection with free MTX. More efficient bone erosion inhibition was observed in mice injected with MTX@HSA NMs, in comparison with treatment of an equivalent dose of MTX. Examination of hematoxylin-eosin (H&E) stained histological slides of ankle joints revealed extensive pannus formation and severe bone destruction in control mice, which translated in the highest histological synovitis score (HSS>8.0) (Figure 6C and 6D).48 Although the mice treated with free MTX showed less extensive cartilage damage, inflammatory cell infiltration and pannus formation were still obvious. Mice injected with MTX@HSA NMs at a dose of 5 mg/kg of MTX per body weight exhibited the lowest HSS values (

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