Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

Manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of T cell response

The mechanism behind the immunostimulatory effect of the cationic liposomal vaccine adjuvant dimethyldioctadecylammonium and trehalose 6,6′- dibehenate (DDA:TDB) has been linked to the ability of these cationic vesicles to promote a depot after administration, with the liposomal adjuvant and the antigen both being retained at the injection site. This can be attributed to their cationic nature, since reduction in vesicle size does not influence their distribution profile yet neutral or anionic liposomes have more rapid clearance rates. Therefore the aim of this study was to investigate the impact of a combination of reduced vesicle size and surface pegylation on the biodistribution and adjuvanticity of the formulations, in a bid to further manipulate the pharmacokinetic profiles of these adjuvants. From the biodistribution studies, it was found that with small unilamellar vesicles (SUVs), 10% PEGylation of the formulation could influence liposome retention at the injection site after 4 days, whilst higher levels (25 mol%) of PEG blocked the formation of a depot and promote clearance to the draining lymph nodes. Interestingly, whilst the use of 10% PEG in the small unilamellar vesicles did not block the formation of a depot at the site of injection, it did result in earlier antibody response rates and switch the type of T cell responses from a Th1 to a Th2 bias suggesting that the presence of PEG in the formulation not only control the biodistribution of the vaccine, but also results in different types of interactions with innate immune cells. © 2012 Elsevier B.V.

Zwitterionic and charge-balanced (polyampholyte) copolymer hydrogels

Zwitterionic compounds, or zwitterions, are electrically neutral compounds having an equal number of formal unit charges of opposite sign. In common polyzwitterions the zwitterionic groups are usually located in pendent groups rather than the backbone of the macromolecule. Polyzwitterions contain both the anion and cation in the same monomeric unit, unlike polyampholytes which can contain the anion and cation in different monomeric units. The use of cationic and anionic monomers (or monomers capable of becoming charged) in stoichiometric equivalent proportions produces charge-balanced polyampholyte copolymers. Hydrogel materials produced from zwitterionic monomers have been proposed for use and are used in many biomaterial applications but synthetic charge-balanced polyampholyte are less common. Certain properties of hydrogels which are important for their successful use as biomaterials, these include the equilibrium water content, mechanical, surface energy, oxygen permeability, swelling and the coefficient of friction. The zwitterionic monomer N,N-dimethyl-N-(2-acryloylethyl)-N-(3-sulfopropyl) ammonium betaine (SPDA) was synthesized with 2-hydroxyethly acrylate (HEMA) as the comonomer to produce a series of polyzwitterion hydrogels. To produce charged-balanced copolymer hydrogels two “cationic” monomers were selected; 2-(diethylamino) ethyl methacrylate (DMAEMA) and 3-(dimethylamino) propyl methacrylamide (DMAPMA) and an anionic monomer; 2-acrylamido 2,2 methylpropane sulphonic acid (AMPS). Two series’ of charge-balanced copolymers were synthesized from stoichiometric equivalent ratios of DMAEMA or DMAPMA and AMPS with HEMA as a terpolymer. The zwitterionic copolymer and both charge-balanced copolymers produced clear, cohesive hydrogels. The zwitterionic and charge-balanced copolymers displayed similar EWC’s along with similar mechanical and surface energy properties. The swelling of the zwitterionic copolymer displayed antipolyelectrolyte behavior whereas the charge-balanced copolymers displayed behaviour somewhere between this and a typical polyelectrolyte. This work describes some aspects of the polymerisation and properties of SPDA copolymers and charge-balanced (polyampholyte) copolymers relevant to their potential as biomedical / bioresponsive materials. The biomimetic nature of SPDA together with its compatibility with other monomers makes it a useful and complimentary addition to the building blocks of biomaterials.

Nanotechnology for the delivery of vaccines

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

Charge-balanced copolymer hydrogels:potential as biomedical materials

Polyzwitterionic-containing hydrogel materials been proposed for use in biomaterial applications. Polyzwitterions contain anions and cations in the same monomeric unit, unlike polyampholytes which contain them in different monomeric units. The use of cationic and anionic monomers in stoichiometrically equivalent proportions produces charge-balanced polyampholytes (PA) copolymers. Membranes prepared using either betaine-containing (BT) polyzwitterionic copolymers or PA copolymers can share similar properties, but the range of EWCs offered by membranes incorporating BT and PA monomers is greater than that for conventional neutral hydrogels and methacrylic acid-based systems. Here we compare properties of BT-containing and PA-containing copolymer membranes, relevant to their potential as biomedical materials. Membranes of the copolymers were prepared as previously described. Surface energy was determined using a GBX Digidrop (GBX Scientific Instruments), with diidomethane and water as probes. The absorption of proteins was determined by soaking the membranes in 1mg/ml protein solutions for a predetermined time, and measuring UV absorption of the membranes at certain wavelengths. The BT and PA copolymer membranes displayed similar values for the polar components and dispersive components of total surface free energy. This was perhaps not surprising when the structures of the monomers were considered. The BT and PA copolymer membranes displayed differences in their protein absorption over time, with the PA demonstrating higher uptake of protein than the BT. In addition to the aforementioned greater EWC range, the use of BT and PA copolymer membranes also avoids some of the problems associated with net anionicity. Comparison of the BT copolymer with the “pseudo” zwitterionic PA copolymers shows that controlled molecular architecture is required to gain the benefits of balancing the charges present in the copolymers in a way that will make them beneficial to hydrogel design.

Liposome (Lipodine™)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine™ liposomes composed of 16 μmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 μmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 μmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1:0.5:0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 μg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 μg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine™) may be a useful system for the oral delivery of DNA vaccines.

Liposome formulation of poorly water soluble drugs:optimisation of drug loading and ESEM analysis of stability

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C 24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 μmol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 μmol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 μmol:4 μmol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 μmol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome. © 2004 Elsevier B.V. All rights reserved.

Liposome-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Preparation and characterisation of amino acids based trimethoprim salts

Trimethoprim (TMP) is a dihydrofolate reductase (DHFR) inhibitor which prevents the conversion of dihydrofolic acid into tetrahydrofolic acid, resulting in the depletion of the latter and leading to bacterial death. Oral bioavailability of TMP is hindered by both its low solubility and low permeability. This study aims to prepare novel salts of TMP using anionic amino acids; aspartic and glutamic acid as counter ions in order to improve solubility and dissolution. TMP salts were prepared by lyophilisation and characterized using FT-IR spectroscopy, proton nuclear magnetic resonance (1HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). Both the amino acids formed salts with TMP in a 1:1 molar ratio and showed a 280 fold improvement in solubility. Investigation of the microbiological activity of the prepared salts against TMP sensitive Escherichia coli showed that the new salts not only retained antibacterial activity but also exhibited higher zone of inhibition which was attributed to improved physicochemical characters such as higher solubility and dissolution. The results are an important finding that could potentially impact on faster onset of antibacterial activity and reduced therapeutic dose when administered to patients. Studies are underway investigating the effect of ion-pairing TMP with amino acids on the permeability profile of the drug.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives - Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6'-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn-glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B–ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods - Liposomal were prepared at a 5?:?1 DDA–TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings - As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion - These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system.

The performance of poly(styrene)-block-poly(2-vinyl pyridine)-block- poly(styrene) triblock copolymers as pH-driven actuators

Poly(styrene)-block-poly(2-vinyl pyridine)-block-poly(styrene) (PS-b-P2VP-b-PS) triblock copolymers were synthesised by anionic polymerisation. Thick films were cast from solution and their structure analysed by small angle X-ray scattering (SAXS). Longer annealing times led to more ordered structures whereas short evaporation times effectively "lock" the polymer chains in a disordered state by vitrification. Well-ordered structures not only provide an isotropic network, which reduces localised stress within the material, but are also essential for fundamental studies of soft matter because their activity on the molecular scale must be analysed and understood prior to their use in technological applications. Well-characterised PS-b-P2VP-b-PS materials have been coupled to a pH-oscillating reaction and their potential application as responsive actuators is discussed. This journal is © The Royal Society of Chemistry.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6′-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn- glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B-ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods Liposomal were prepared at a 5: 1 DDA-TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system. © 2013 Royal Pharmaceutical Society.

CO-stabilisation mechanisms of nanoparticles and surfactants in Pickering emulsions produced by membrane emulsification

Two different membrane emulsification methods were used to study mechanisms for co-stabilisation of emulsions, by either electrostatic or steric stabilised nanoparticles with anionic, cationic or non-ionic surfactants. The experimental results demonstrated the existence of two distinct co-stabilisation mechanisms that arise from interactions of the nanoparticles and surfactant molecules. When significant interaction is not involved, independent competitive adsorption of nanoparticles and surfactant molecules occurs spontaneously to stabilise droplets in formation. The adsorption/desorption equilibrium between surfactant molecules determines the longevity of the droplet stability. When the surfactant molecule reacts with the nanoparticle surface, the resultant surface modification appears to generate faster wetting kinetics for nanoparticles at the oil/water interface and yields enhanced stabilisation. The paper discusses the implications of controlling these interactions for emulsion production membrane systems.

Photoluminescent materials based on PMMA and a highly-emissive octahedral molybdenum metal cluster complex

Materials that combine photoluminescence, optical transparency and facile processability are of high importance in many applications. This article reports on the development of photoluminescent poly(methyl methacrylate) materials based on novel highly emissive anionic molybdenum cluster complex [{Mo6I8}(OTs)6]2– (where OTs– is the p-toluenesulfonate ion). The materials were obtained by both solution and bulk copolymerisation of methyl methacrylate and (dMDAEMA)2[{Mo6I8}(OTs)6], where dMDAEMA+ is the polymerisable cation [2-(methacryloyloxy)ethyl]dimethyl-dodecylammonium. Evaluation of the resultant hybrid materials showed that one could combine the excellent photoluminescent properties of the cluster complex with the transparency and processability of PMMA.

Immunomodulation by shark cartilage extracts

The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-α) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-á could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-κB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.