High MW Hyaluronan Research Articles

Development of a drying method for proteins based on protein-hyaluronic acid precipitation

This study aims to develop a new method to dry proteins based on protein-hyaluronic acid (HA) precipitation and apply the precipitation-redissolution technique to develop highly concentrated protein formulations. Lysozyme was used as a model protein and HA with various molecular weights (MW) were investigated. Under low ionic strength, low-MW HA (e.g., MW: around 5 K) did not induce lysozyme precipitation. Conversely, high-MW HA (e.g., MW: approximately from 40 K to 1.5 M) precipitated more than 90 % of lysozyme. The dried lysozyme-HA precipitates had moisture levels between 4 % and 5 %. The lysozyme-HA precipitates could be redissolved using PBS (pH 7.4, ionic strength: ∼ 163 mM). The viscosity of the reconstituted solution was dependent on HA MW, e.g., 4 cP for HA40K, and 155 cP for HA1.5 M, suggesting low-MW HA might be a proper excipient for highly concentrated solution formulations for subcutaneous/intraocular injection and high-MW HA may fit for other applications. The tertiary structure of lysozyme after the precipitation-redissolution steps had no significant difference from that of the original lysozyme as confirmed by fluorescence spectroscopy. The denaturation temperature of lysozyme after the precipitation-redissolution steps and that of the original lysozyme were close, indicating they possessed similar thermal stability. The accelerated stability study revealed that lysozyme stored in the dry precipitates was more physically stable than that in the buffer solution. Overall, this new drying technique is suitable for drying proteins and exhibits several benefits such as minimum energy consumption, cost effectiveness, high production yield, and mild processing conditions. In addition, the precipitation-redissolution technique proposed in this study can potentially be used to develop highly concentrated formulations, especially for proteins experiencing poor stability in the liquid state.

Injecting hyaluronan in the thoracolumbar fascia: A model study

Hyaluronan (HA) has been recently identified as a key component of the densification of thoracolumbar fascia (TLF), a potential contributor to non-specific lower back pain (LBP) currently treated with manual therapy and systemic or local delivery of anti-inflammatory drugs. The aim of this study was to establish a novel animal model suitable for studying ultrasound-guided intrafascial injection prepared from HA with low and high Mw. Effects of these preparations on the profibrotic switch and mechanical properties of TLF were measured by qPCR and rheology, respectively, while their lubricating properties were evaluated by tribology. Rabbit proved to be a suitable model of TLF physiology due to its manageable size enabling both TLF extraction and in situ intrafascial injection. Surprisingly, the tribology showed that low Mw HA was a better lubricant than the high Mw HA. It was also better suited for intrafascial injection due to its lower injection force and ability to freely spread between TLF layers. No profibrotic effects of either HA preparation in the TLF were observed. The intrafascial application of HA with lower MW into the TLF appears to be a promising way how to increase the gliding of the fascial layers and target the myofascial LBP.

MP44-10 HIGH MOLECULAR WEIGHT HYALURONAN ATTENUATES SCARRING IN ACUTE KIDNEY INJURY

You have accessJournal of UrologyPediatric Urology II (MP44)1 Sep 2021MP44-10 HIGH MOLECULAR WEIGHT HYALURONAN ATTENUATES SCARRING IN ACUTE KIDNEY INJURY Pratik Kanabur, Aditya Kaul, Scott Manson, Swathi Balaji, Paul Austin, Sandeep Keswani, and Xinyi Wang Pratik KanaburPratik Kanabur More articles by this author , Aditya KaulAditya Kaul More articles by this author , Scott MansonScott Manson More articles by this author , Swathi BalajiSwathi Balaji More articles by this author , Paul AustinPaul Austin More articles by this author , Sandeep KeswaniSandeep Keswani More articles by this author , and Xinyi WangXinyi Wang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002065.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal fibrosis induces morbidity in pediatric patients, which can persist into adulthood. Hyaluronan (HA), a major extracellular matrix component, regulates fibrosis based on its molecular weight (MW). We have reported that IL-10 upregulates high MW-HA synthesis via fibroblasts to attenuate fibrosis in a chronic kidney disease model. However, the effect of high MW-HA in acute kidney injury is unknown. Our data suggests that HA is upregulated in epithelial tubular cells in an acute kidney injury model. We hypothesize that tubular cell-specific high MW-HA reduces fibrosis caused by acute kidney injury. METHODS: We performed unilateral ischemia/reperfusion(I/R) on C57BL/6J male mice treated with and without IL-10 and also with and without 4-methylumbelliferone(4MU), an HA inhibitor. I/R or sham kidneys were collected at Day 3, 7, and 14 for the levels of HA synthases(HAS1-3) and hyaluronidases(HYAL1-2) by qPCR, total HA by ELISA, and fibrosis outcome and tubular cell proliferation by immunohistochemistry. In vitro studies were also done with human proximal tubular HK-2 cells, which were used to determine the effect of IL-10 on HA enzymes with/without TGFβ, which mimics the fibrotic phenotype. ANOVA and Student’s t-test were used as appropriate with statistical significance defined as p<0.05. RESULTS: I/R significantly increases HA production with the peak of enzyme production at day 3. With IL-10 treatment, we observe upregulated total HA at D3&D7, increased tubular cell proliferation, and reduced fibrosis. These effects however are not observed when HA is inhibited by 4-MU. In vitro, TGFβ significantly alters expression of HAS1&3 and HYAL1 compared to control, and IL-10 treatment increases HA and restores HAS1 level compared to control (all p<0.05) CONCLUSIONS: Tubular-specific HA plays a protective role in response to acute renal injury, and the upregulation of HA by IL-10 can attenuate fibrosis. We identified a previously unreported role for high MW-HA that occurs in response to kidney injury. We also elucidated the mechanisms underlying HA-attenuated fibrosis to inspire novel therapeutics. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e796-e796 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pratik Kanabur More articles by this author Aditya Kaul More articles by this author Scott Manson More articles by this author Swathi Balaji More articles by this author Paul Austin More articles by this author Sandeep Keswani More articles by this author Xinyi Wang More articles by this author Expand All Advertisement Loading ...

Predicting intra-articular residence time of hyaluronan-based viscosupplements. a comparison between human and artificial synovial fluid as degradation media

Purpose: Hyaluronan(HA)-based viscosupplements, although resorbable, present different in vivo residence times, which are mainly related to structural modifications of the native polymer. The residence time of unmodified HA generally does not exceed 5 days, while chemically crosslinked HA can be detected in the joint up to one month. Artificial synovial fluid (ASF) have been described in the literature to mimic rheology of or drug release in human SF (SF). These models suffer from a limitation from the biological point of view: the absence of hyaluronidases, HA degrading enzymes expressed by the chondrocytes in contact with SF, well described in literature. A novel in vitro preparation of ASF with hyaluronidase is hereby reported and tested with three marketed viscosupplements: Sinovial® HL (SHL, IBSA Farmaceutici), which contains native HA of both low and high MW, Hyalubrix® (HBX, Fidia Farmaceutici S.p.A.), which contains unmodified high MW HA and Hymovis® (HYM, Fidia Farmaceutici S.p.A.), composed of HA modified with hexadecyl alkyl chains via amidic bonds (MW comprised between 500 and 700 kDa). The effect of this ASF towards HA molecular weight and rheological properties was reported and compared with the degradation profile in centrifuged human SF. Methods: ASF was formulated with the following composition: NaCl 8 g/L, KCl 0.2 g/L, Na2HPO4 1.7 g/L, Bovine Serum Albumin (BSA) 19 g/L, Bovine Testes Hyaluronidase (BTH) 6 U/mL, Gentamicin 0.01 mg/mL (pH 7.4). The salt and BSA composition and concentration in SF are well reported in the literature. At the best of our knowledge, the quantification of hyaluronidase activity in human SF is not reported. Considering that Hyaluronidase (Hyal) activity titrated in serum is at least 6 U/mL in BTH equivalent, and that Hyal (MW 55-60 kDa) can permeate synovial membrane, the same concentration of BTH was added to artificial SF. Centrifuged (cell free) human SF collected from OA patients was used as “degradation media”. Sinovial® HL, Hyalubrix® and Hymovis® were separately incubated either in ASF or SF at 37 °C for 14 days at a w/w ratio of approximately 1:1, under mild shaking. At different time points (0, 1, 7 and 14 days) the viscoelastic moduli and dynamic viscosity of the mixtures were measured with an Anton Paar MCR 92 rheometer. Moreover, at each time point the mixtures were diluted in water and filtered through Merck Vivaspin® 2 centrifugal concentrators (MWCO = 100 kDa, comparable to the synovial membrane cut-off) in order to quantitate the permeated and retained HA fractions. PBS+SF at a w/w ratio of 1:1 was used as blank for HA measurement. The percent recovery of the HA fraction with MW ≤ 100 kDa was determined by SEC-TDA (Omnisec Resolve/Reveal, Malvern instruments). Results: The degradation profile of HYM, HBX and SHL in SF or ASF is shown in the graph below: after 14 days the viscoelasticity (expressed as storage modulus: G’) of HYM/SF, HBX/SF and SHL/SF is slightly decreased; however, the elasticity of all viscosupplements in ASF is completely impaired after only few hours. After 14 days, the percent recovery of high MW HA (>100 kDa) confirms the rheological data: in SF, HYM, HBX and SHL were not depolymerized to low MW polymers while, in ASF, the percent recovery in higher MW HA was only about 45%, 10% and 13% for HYM, SHL and HBX respectively. The strong depolymerizing activity of ASF was confirmed for all the viscosupplements tested; nonetheless, HYM showed higher resistance to degradation in ASF compared to SHL. Conclusions: In this work, two different in vitro and ex vivo models were compared in order to predict the in vivo residence time of three HA-based viscosupplements in the intra-articular environment. Based on the literature, after a single intra-articular administration of high MW HA (2000 kDa), the measured residence time was as short as about 5 days. The ex vivo model with centrifuged human SF bears the limitation of the absence of hyaluronidases (a cell surface glycosylphosphatidylinositol-anchored protein, removed with the chondrocytes during the centrifugation process), while the assay in ASF added with BTH turned out to be more reliable and can therefore be employed during viscosupplement development to predict the intra-articular residence time of HA-based products

Effects of Hyaluronan Molecular Weight on the Lubrication of Cartilage-Emulating Boundary Layers.

Osteoarthritic joints contain lower-molecular-weight (MW) hyaluronan (hyaluronic acid, HA) than healthy joints. To understand the relevance of this HA size effect for joint lubrication, the friction and surface structure of cartilage-emulating surfaces with HA of different MWs were studied using a surface force balance (SFB) and atomic force microscopy (AFM). Gelatin (gel)-covered mica surfaces were coated with high-MW HA (HHA), medium-MW HA (MHA), or low-MW HA (LHA), and lipids of hydrogenated soy l-α-phosphatidylcholine (HSPC) in the form of small unilamellar vesicles, using a layer-by-layer assembly method. SFB results indicate that the gel-HHA-HSPC boundary layer provides very efficient lubrication, attributed to hydration lubrication at the phosphocholine headgroups exposed by the HA-attached lipids, with friction coefficients (COF) as low as 10–3–10–4 at contact stresses at least up to P = 120 atm. However, for the gel-MHA-HSPC and gel-LHA-HSPC surfaces, the friction, initially low, increases sharply at much lower pressures (up to 30–60 atm at most). This higher friction with the shorter chains may be due to their weaker total adhesion energy to the gelatin, where the attraction between the negatively charged HA and the weakly positively charged gelatin is attributed largely to counterion-release entropy. Thus, the complexes of LHA and MHA with the lubricating HSPC lipids are more easily removed by shear during sliding, especially at high stresses, than the HHA-HSPC complex, which is strongly adhered to gelatin. This is ultimately the reason for lower-pressure lubrication breakdown with the shorter polysaccharides. Our results provide molecular-level insight into why the decrease in HA molecular weight in osteoarthritic joints may be associated with higher friction at the articular cartilage surface, and may have relevance for treatments of osteoarthritis involving intra-articular HA injections.

Modulating Three-Dimensional Microenvironment with Hyaluronan of Different Molecular Weights Alters Breast Cancer Cell Invasion Behavior.

Hyaluronan (HA), a polymer with various molecular weights (MW) found in tumor microenvironments, is associated with malignant progression of breast cancer. Reducing the amount of high-MW HA in the microenvironment by hyaluronidase is a promising approach for breast cancer treatment. However, whether the generation of HA fragments negatively affects breast cancer cells remains to be determined. Furthermore, HA forms three-dimensional (3D) networks by cross-linking with other extracellular molecules to function. Therefore, a model mimicking the cross-linked HA network is required to determine the effect of HA fragments on breast cancer cells. To clarify the differential roles of low (HA35) versus high (HA117) MW HA on cancer cell phenotype, a 3D culture system was set up by covalently cross-linking HA with alginate and investigating the behavior of 4T-1 and SKBR3 breast cancer cells alongside a two-dimensional (2D) control. The results show the invasion and migration abilities of 4T-1 and SKBR3 cells are significantly enhanced by the presence of HA35 but inhibited by HA117 in both 2D monolayers and 3D spheroids. The differential effects of HA35 and HA117 on cancer cell epithelial-mesenchymal transition (EMT) phenotype were further confirmed in terms of differential regulation of E-cadherin and vimentin as important EMT markers at both the cellular and mRNA levels. Additional experiments show the CD44-Twist signaling pathway might be involved in the differential effects of HA35 and HA117. These results have important implications with respect to understanding the role of HA in breast cancer development and for the design of therapeutic approaches based on the eradication of HA with hyaluronidase.

Is molecular size a discriminating factor in hyaluronan interaction with human cells?

Nowadays there is a great interest in investigating the effect of particular hyaluronan fragments in the biomedical field and in cosmeceutical applications. Literature has reported that very low molecular weight HA (Mw<5kDa) has an inflammatory effect, whilst HA ranging from 15 to 250 has shown controversial effects. This work aims to give better elucidation on the correlation between the different sized HA fragments and their biological functions. In this respect, a simple and effective degradation strategy is used to obtain several HA fragments. Also, an hydrodynamic and structural characterization was performed in order to obtain samples suitable to evaluate cellular response. In particular an in vitro scratch test in time lapse experiments was used to study the effect of HA fragments, ranging from 1800 to 6kDa on wound dermal reparation based on human keratinocytes. All high and low Mw HA used in this study allowed for faster wound closure compared to the un-treated cells, except for 6kDa that, on the contrary, prevented repair.In addition, TGF-β 1, TNFα and IL-6, representative biomarkers of the inflammation phase occurring in wound healing process, were quantified by RT-PCR. A general up-regulation trend of these biomarkers was found with the HA molecular weight reduction. LHA6kDa was the only treatment that induced a major inflammatory response (over 30 fold increase respect to control) confirming the recent literature outcomes. IL-6 protein level evaluated through ELISA assay corroborated the previous results. Furthermore, activation of key HA receptors, such as CD44, RHAMM, TLR4, with respect to hyaluronan size, was evaluated, at transcriptional level showing selective recognition by HA 1800, 1400, 500 for CD44, whilst the lower Mw fragments activated TLR-4 moderately at 50 and 15kDa. An increase to “alarm” level was found for 6kDa fragments. Immunofluorescence staining confirmed this data.The present research work demonstrated that the diverse pharma grade hyaluronan fragments could modulate cellular processes differently. From 1800kDa down to 50kDa, CD44 was the recognized receptor and pro-inflammatory biomarkers were only slightly up-regulated during wound healing in the presence of HA. Finally our outcomes showed that the lower the fragment size the higher the concern for inflammatory cytokines up-regulation; repair process impairment was highlighted only for 6kDa chains.

Selective Targeting and Restrictive Damage for Nonspecific Cells by Pulsed Laser-Activated Hyaluronan-Gold Nanoparticles.

Herein, we describe an approach that immobilizes low-molecular-weight hyaluronic acid (low-MW HA) on the surface of gold nanoparticles (GNPs), which can serve as a cellular probe and photodamage media, to evaluate the selectivity and efficiency of HA-based GNPs (HGNPs) as a mediator of laser-induced photothermal cell damage. In addition, it is known that solid tumors contain a higher content of low-MW HA than normal tissues. Thus, we used low-MW HA rather than high-MW HA used in other studies. In the present study, we conjugated low-MW HA, which is a linear polysaccharide with a disaccharide repeat unit, to prevent a reduction of the ligand-receptor binding efficiency in contrast to the conjugation of protein or peptides, which have unique three-dimensional structures. Three cell lines-MDA-MB-435 S (with CD44), MDA-MB-453 and NIH/3T3 (both are without CD44)-were investigated in the study, and qualitative observations were conducted by dark-field microscopy and laser scanning confocal microscopy (LSCM). In addition, quantitative measurements calculated using inductively coupled plasma emissions were taken for comparison. Our results showed that within the same treatment time, the uptake dosage of HGNPs by the MDA-MB-435 S cells was higher than that by the MDA-MB-453 and NIH 3T3 cells. Meanwhile, HGNPs uptake by the untreated MDA-MB-435 S cells was higher than that of MDA-MB-435 S cells with CD44 blocked by antibodies or silencing CD44 expression. This result implies that receptor-mediated endocytosis can enhance the cellular uptake of HGNPs. In addition, when exposed to a low-power pulsed laser, the former cell morphologies showed a more laser-induced giant plasma membrane vesicles (GPMV) than the latter morphologies. Therefore, this study utilized the specific photothermal property of HA-modified GNPs with laser-induced blebs to create a possible new method for medical applications.

Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover.

We studied the physiologic roles of the hyaluronidase (HYAL) 1 and HYAL2 in hyaluronan (HA) turnover. HA was localized and quantified using HA binding proteins in various tissues of Hyal1(-/-) and Hyal2(-/-) mice (knockout mice) as well as control mice. HA MW was determined using gel filtration chromatography. HA endocytosis in liver nonparenchymal cells (NPCs) was quantified in vivo Both Hyal1 and Hyal2 knockout mice showed HA accumulation in peripheral tissues without changes in HA MW distribution. HYAL2 deficiency induced buildup of very high MW (>3.10(6) Da) HA in lymph and serum with severe lymph node distortion. The lack of HYAL2 also impaired high MW HA endocytosis by liver NPCs. HYAL1 deficiency led to a moderate increase in serum HA concentration without changes in HA MW distribution and to HA overload of liver NPCs. Wild-type C57BL/6 mice served as controls. In HA injection experiments, saline-injected mice served as additional controls. We conclude that: 1) HYAL1 and HYAL2 are both needed for tissue HA catabolism; 2) HYAL2 is required for high MW HA clearance in lymph nodes and plasma and for HA endocytosis by liver NPCs; and 3) the main role of HYAL1 is HA degradation within liver NPCs.-Bourguignon, V., Flamion, B. Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover.

Cartilage boundary lubrication synergism is mediated by hyaluronan concentration and PRG4 concentration and structure

BackgroundProteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4.MethodsStatic and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively.ResultsThis study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important.ConclusionsCollectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.

Intracellular hyaluronan protects murine fibroblasts against apoptosis (1003.3)

Skin repair typically involves apoptotic cell death. We are interested in the role of hyaluronan (HA), an extracellular matrix molecule, during fibroblast‐mediated repair of injured skin. Has1/3 null mice (Has1/3), which lack hyaluronan synthases 1 and 3 (yet retain Has2) show accelerated wound closure, more fibroblasts, and fewer cleaved caspase‐3–positive dermal cells in healing wounds, suggestive of reduced apoptosis. In cultured Has1/3 fibroblasts, HA levels and Has2 mRNA expression were higher than in WT cells. Apoptosis, induced by serum starvation (SS) or UVB exposure, was reduced in the Has1/3 cells as shown by DNA fragmentation and by cleavage of caspases and PARP. Hyaluronidase, added to remove extracellular HA, surprisingly showed no effect on apoptotic susceptibility to SS or UVB. However, cells treated with 4‐MU to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. To further establish a role for HA in protection from apoptosis, Has2 RNA interference (RNAi) was done using Has2‐specific siRNA transfection. Has2 RNAi in WT and Has1/3 fibroblasts inhibited HA synthesis by 90% and significantly increased their sensitivity to apoptosis. External addition of high MW HA failed to reverse this effect. We conclude that Has1/3 skin fibroblasts (which make relatively more HA) are more resistant to apoptosis due to the presence of intracellular HA synthesized by Has2.Grant Funding Source: supported by NHLBI

AB0998 Medium and high molecular weight hyaluronic acid injected in hip joint by US-guided technique in patients with primary or secondary hip osteoarthritis: A 2 year observational study

BackgroundGuidelines for treatment of osteoarthritis (OA) of the hip of the American College of Rheumatology provide intra-articular therapy with steroids and hyaluronic acid (HA) in addition to standard therapy. The...

The visk study: a cost utility analysis of intra articular hyaluronic acid for knee osteoarthritis

usual care for more than 6 months were included. Patients were randomized into a control group receiving usual care or into an intervention group receiving usual care with 3 additional intra articular injections with high MW HA. Data on pain during rest / activity, function and global assessment were collected during 12 months follow-up. The primary outcome was defined as response to therapy according to OMERACT-OARSI criteria (improvement of at least 20% in at least 2 of the 3 following domains: pain in rest / pain during activity, function and patients global assessment) after 12 months of follow-up. Adverse events were registered during follow-up. Results: In total, 156 subjects were included of which 77 patients in the intervention group and 79 subjects in the control group. There were statistically significant more responders in the intervention groupwhen pain during rest was included in responder criteria (51.9% versus 26.7%, p1⁄40.001) after 12 months. With pain during activity as responder criteria, this difference was also statistically significant favouring the intervention group (51.9% versus 25.3%, p1⁄40.001). Also on the individual outcomes of the responder criteria significant differences were found between both groups favouring the intervention group. Conclusions: Intra articular HA added to usual care leads to significantly more responders according to OMERACT-OARSI criteria after 12 months.

Hyaluronan-coated nanoparticles: The influence of the molecular weight on CD44-hyaluronan interactions and on the immune response

Hyaluronan (HA), a naturally occurring glycosaminoglycan, exerts different biological functions depending on its molecular weight ranging from 4000–10M Da. While high Mw HA (HMw-HA) is considered as anti-inflammatory, low Mw HA (LMw-HA) has been reported to activate an innate immune response. In addition, opposing effects on cell proliferation mediated by the HA receptor CD44, have also been reported for high and low Mw HA. We have previously demonstrated that HMw-HA can be covalently attached to the surface of lipid nanoparticles (NPs), endowing the carriers with long circulation and active targeting towards HA-receptors (CD44 and CD168) highly expressed on tumors. Here we present a small library of HA-coated NPs distinguished only by the Mw of their surface anchored HA ranging from 6.4 kDa to 1500 kDa. All types of NPs exerted no effect on macrophages, T cells and ovarian cancer cells proliferation. In addition, no induction of cytokines or complement activation was observed. The affinity towards the CD44 receptor was found to be solely controlled by the Mw of the NPs surface-bound HA, from extremely low binding for LMw-HA to binding with high affinity for HMw-HA. These findings have major implications for the use of HA in nanomedicine as LMw-HA surface modified-NPs could be a viable option for the replacement of polyethylene glycol (PEG) when passive delivery is required, lacking adverse effects such as complement activation and cytokine induction, while HMw-HA-coated NPs could be used for active targeting to CD44 overexpressing tumors and aberrantly activated leukocytes in inflammation.

High MW hyaluronan inhibits smoke inhalation‐induced lung injury and improves survival

High MW hyaluronan (HMW HA) as opposed to low MW hyaluronan (LMW HA) has been shown to have anti-inflammatory and anti-apoptotic effects. We hypothesized that treatment with HMW HA would block smoke inhalation lung injury by inhibiting smoke-induced lung inflammation and airway epithelial cell apoptosis. Anesthetized, intubated male rats were randomly allocated to either control or smoke inhalation injury groups. Rats were treated with 3-mL subcutaneous normal saline solution (sham) or LMW HA (35 kDa) or HMW HA (1600 kDa) 18 h before exposure to 15 min of cotton smoke (n = 5 each). Rats were also treated post smoke inhalation with 1600 kDa HA by intra-peritoneal injection (3 mL) or intra-tracheal nebulization (200 µL). Lung neutrophil infiltration, airway apoptosis, airway mucous plugging and lung injury were assessed 4 h after smoke inhalation injury. Rats pretreated with 1600 kDa HA had significantly less smoke-induced neutrophil infiltration, lung oedema, airway apoptosis and mucous plugging. Pretreatment with 35 kDa HA, in contrast, increased smoke-induced neutrophil infiltration and lung injury score. Intra-tracheal administration of a single dose 1600 kDa HA, but not intra-peritoneal injection, significantly improved survival post smoke inhalation. High MW hyaluronan (1600 kDa) may prove to be a beneficial therapy for smoke inhalation through inhibition of smoke-induced inflammation, lung oedema, airway epithelial cell apoptosis and airway mucous plugging.

Semi‐permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi-permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 microm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes +/- IL-1beta + TGF-beta1 + TNF-alpha, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes +/- adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 microg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine-regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 microm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was approximately 20 x 10(-8) cm/s (- cells) and approximately 5 x 10(-8) cm/s (+ cells), for 90 nm membranes was approximately 35 x 10(-8) cm/s (- cells) and approximately 19 x 10(-8) cm/s (+ cells), for 170 nm membranes was approximately 74 x 10(-8) cm/s (+/- cells), and for 3 microm membranes was approximately 139 x 10(-8) cm/s (+/- cells). The permeability of 450 kDa HA was approximately 40x lower than that of 30 kDa HA for 50 nm membranes, but only approximately 2.5x lower for 3 microm membranes. The permeability of 4,000 kDa HA was approximately 250x lower than that of 30 kDa HA for 50 nm membranes, but only approximately 4x lower for 3 microm membranes. The permeability for PRG4 was approximately 4 x 10(-8) cm/s for 50 nm membranes, approximately 48 x 10(-8) cm/s for 90 nm membranes, approximately 144 x 10(-8) cm/s for 170 nm membranes, and approximately 336 x 10(-8) cm/s for 3 microm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi-permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for lubricant transport.

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) –mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-α and IL-1β levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation.

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

Background Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes. Methods mRNA and related protein levels were measured for CD44, PKCδ, PKCɛ, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed. Results CD44, PKCδ, and PKCɛ mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKCɛ levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKCɛ, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKCɛ, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation. General Significance These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.

Regulation of polyurethane hemocompatibility and endothelialization by tethered hyaluronic acid oligosaccharides

Current synthetic vascular grafts possess a significant mechanical mismatch compared to the native vasculature and do not permit endothelialization; both of these deficiencies contribute to the relatively high rate of failure of many synthetic grafts. In this communication, we report the modification of polyurethane (PU)-based materials to impart hemocompatibility, support endothelial growth, and display vascular-appropriate mechanics. This modification was achieved by incorporating branched polyethylenimine (PEI) into the PU backbone, followed by covalent attachment of either hyaluronic acid (HA; 4.7, 64, and 104 kDa), heparin, or poly(ethylene glycol) (PEG; used as a non-adhesive control) to the PEI. This grafting chemistry resulted in comparatively dense immobilization of HA and heparin (0.062 and 2.3 μg/cm 2, respectively) to the PU–PEI surfaces. PU materials modified with HA were more effective than either PEG- or heparin-grafted materials with respect to limiting protein adsorption and platelet adhesion. Confluent, morphologically-healthy cultures of endothelial cells were achieved only on materials grafted with low molecular weight HA, but not high MW HA, heparin, or PEG. These modifications in PU chemistry were performed while retaining material mechanics in the range of native vascular tissue. Thus, this study describes the generation of materials that possess the unique ability to display excellent hemocompatibility while simultaneously supporting extensive endothelialization and retaining vascular-appropriate mechanics. The bioactivity of these materials was regulated by the molecular weight of the grafted HA, and their physical and biological properties make them promising for use as vascular grafts.

A reappraisal of the biological effects of hyaluronan on human dermal fibroblast

Hyaluronan (HA) is a fundamental component of many biomaterials. Reports on the biological effects of HA are, however, both inconsistent and contradictory. This particularly refers to fibroblast contraction in the presence of high concentrations of HA. In this study we used a broad range of molecular weight (MW) and concentrations of HA to assay its influence on human dermal fibroblasts in both two and three-dimensional culture models. High-MW HA in high concentrations demonstrated inhibitory effects on both cell attachment and proliferation. HA-coated surfaces appeared less adhesive to fibroblasts than collagen-coated surfaces. In the fibroblast-populated collagen lattice, HA supplementation dose-dependently retarded gel contraction, which was associated with reduced cell division and spreading. Although gene transcripts of collagen type I and III were not significantly upregulated in the monolayer culture as determined by real-time PCR analysis, more type III collagen was present in HA-incorporated collagen gels by Sirius red staining. Type III collagen was homogeneously distributed and well organized in the presence of HA. Our data indicates that HA has both a qualitative and quantitative effect on cell-matrix interactions. Optimum parameters in terms of MW and concentration need to be determined when HA-based biomaterials are being prepared for tissue-repair purposes.