Design and Synthesis of Nonionic Copolypeptide Hydrogels with Reversible Thermoresponsive and Tunable Physical Properties

Polypeptide-based formulations that undergo liquid to hydrogel transitions upon change in temperature have become desirable targets since they can be mixed with cells or injected into tissues as liquids, and subsequently transform into rigid scaffolds or depots. Such materials have been challenging to prepare using synthetic polypeptides, especially when reversible gelation and tunable physical properties are desired. Here, we designed and prepared new nonionic diblock copolypeptide hydrogels (DCH) containing hydrophilic poly­(γ-[2-(2-methoxyethoxy)­ethyl]-<i>rac</i>-glutamate) and hydrophobic poly­(l-leucine) segments, named DCH_EO, and also further incorporated copolypeptide domains into DCH_EO to yield unprecedented thermoresponsive DCH, named DCH_T. Although previous attempts to prepare nonionic hydrogels composed solely of synthetic polypeptides have been unsuccessful, our designs yielded materials with highly reversible thermal transitions and tunable properties. Nonionic, thermoresponsive DCH_T were found to support the viability of suspended mesenchymal stem cells in vitro and were able to dissolve and provide prolonged release of both hydrophilic and hydrophobic molecules. The versatility of these materials was further demonstrated by the independent molecular tuning of DCH_T liquid viscosity at room temperature and DCH_T hydrogel stiffness at elevated temperature, as well as the DCH_T liquid to hydrogel transition temperature itself

STAT3 Activation does not occur in non-damaged brain regions in multiple models of neurotoxicity.

<p>Mice were administered METH, KA or TMT with saline (0.9%) as a control and were killed by focused microwave irradiation at 12 hrs. post dosing and by decapitation at 72 hours post dosing. Multiple “non-target” brain regions for each neurotoxicant were sampled for activated STAT3 (12 hours post dosing) and levels of GFAP (72 hours post dosing). All data points represent mean ± SEM, N = 5. Statistical significance was measured by one-way ANOVA. Where significant differences were found, Fisher's LSD Method of post hoc analysis was performed. Statistical significance of at least p<0.05 for the neurotoxicant exposed groups in comparison to saline controls is denoted by *. See Methods for dosing regimen (multi-dose METH).</p

Enhanced expression of proinflammatory ligands results from neurotoxicity-induced damage: evidence from neuroprotective agents.

<p>Mice were administered MPTP, METH, or KA alone or after pretreatment with nomifensine (for MPTP), ethanol (for METH) or diazepam (for KA). Mice were killed at 12 hours post dosing. Total RNA was prepared from striatum (MPTP and METH) or hippocampus (KA) for qRT-PCR analysis of <i>Tnf-α</i>, <i>Osm</i>, <i>Ccl2</i> or <i>Lif</i>. All data points represent mean ± SEM, N = 5. * denotes statistical significance of at least p<0.05 for the neurotoxicant alone or pretreated groups compared to saline controls and # denotes statistical significance of at least p<0.05 for neuroprotectant pretreated groups compared to neurotoxicant only exposed groups. Statistical significance was measured by two-way ANOVA with Fisher's LSD Method of post hoc analysis. See Methods for dosing regimen (single dose METH). Data for <i>Osm</i> and <i>Lif</i> in the MPTP groups was taken from Fig. 8 in Sriram et al., (2004) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102003#pone.0102003-Sriram1" target="_blank">[12]</a>.</p

Anti-inflammatory treatment with CORT does not suppress activation of STAT3, GFAP expression, or neurotoxicity.

<p>Mice were administered MPTP, METH, or TMT alone or after pretreatment with CORT (20 mg/kg, s.c.) 30 minutes earlier and killed at the post-dosing times indicated. Mice were killed by focused microwave irradiation to preserve steady-state phosphorylation of pSTAT3^{tyr 705} and striatal phosphorylation was analyzed by quantification of scans of pSTAT3^tyr705 immunoblots. Representative immunoblot data from two different animals for each dosing groups are presented above the quantitative data obtained from scans. Separate groups of mice killed by decapitation were used to prepare total striatal protein homogenates for ELISA of GFAP and TH. All data points represent mean ± SEM, N = 5. * denotes statistical significance of at least p<0.05 for the neurotoxicant alone or CORT pretreated groups compared to saline controls and # denotes statistical significance of at least p<0.05 for CORT- pretreated groups compared to neurotoxicant only exposed groups. Statistical significance was measured by two-way ANOVA with Fisher's LSD Method of post hoc analysis. See Methods for dosing regimens (single dose METH).</p

LPS enhances expression of proinflammatory ligands, CORT-suppressible activation of STAT3, but not expression of GFAP.

<p>Mice were administered LPS (2 mg/kg, s.c.), CORT (20 mg/kg) or CORT (20 mg/kg) 30 minutes prior to LPS (2 mg/kg). Mice were killed at the post-dosing times indicated or at 6 hours post dosing for the LPS and CORT/LPS groups analyzed for pSTAT3^{tyr 705}. Mice were killed by focused microwave irradiation to preserve steady-state phosphorylation of pSTAT3^{tyr 705} and striatal phosphorylation was analyzed by quantification of scans of pSTAT3^tyr705 immunoblots. Separate groups of mice were used to prepare total striatal RNA from one side of the brain for qRT-PCR analysis of <i>Tnf-α</i>, <i>Osm</i>, <i>Ccl2</i> or <i>Lif</i> and to prepare total striatal protein homogenates from the other side of the brain for ELISA of GFAP. All data points represent mean ± SEM, N = 5. * denotes statistical significance of at least p<0.05 for the LPS alone and CORT/LPS groups compared to saline and # denotes statistical significance of at least p<0.05 for CORT- pretreated LPS group compared to LPS only exposed group. Statistical significance was measured by one or two-way ANOVA. Where significant differences were found, Fisher's LSD Method of post hoc analysis was performed. See Methods for other details on dosing regimens.</p

STAT3 activation precedes GFAP up regulation in multiple models of striatal neurotoxicity.

<p>Mice were administered dopaminergic neurotoxicants, MPTP, AMP, METH, MDA and MDMA with saline (0.9%) as a control and were killed at 6, 12 and 72 hours post dosing, time points known to encompass the onset of dopaminergic neurotoxicity and the subsequent activation of microglia and astroglia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102003#pone.0102003-Sriram1" target="_blank">[12]</a>. Mice were killed by focused microwave irradiation to preserve steady-state phosphorylation of pSTAT3^{tyr 705} and phosphorylation was analyzed by quantification of scans of pSTAT3^tyr705 immunoblots. Representative immunoblot data from two different animals killed at each time point are presented above the quantitative data obtained from scans and are denoted by a bracket. Separate groups of mice were used to prepare total RNA from one side of the brain for qRT-PCR analysis of <i>Gfap</i> mRNA and to prepare total protein homogenates from the other side of the brain for ELISA of GFAP and TH. Except for the representative immunoblot duplicates, all data points represent mean ± SEM, N = 5. Statistical significance was measured by one-way ANOVA with Fisher's LSD Method of <i>post hoc</i> analysis. Statistical significance of at least p<0.05 for the neurotoxicant exposed groups in comparison to saline controls is denoted by *. See Methods for dosing regimen (multi-dose METH).</p

Pro-inflammatory treatment with peripherally injected LPS does not result in increased levels of GFAP at 24 h.

<p>Mice were administered LPS (1–5 mg/kg, i.p.) and killed at 24 hours post dosing. Total brain region homogenates were assayed for GFAP by ELISA. All data points represent mean ± SEM, N = 5. Statistical significance was measured by one-way ANOVA. See methods for dosing regimen.</p

STAT3 Activation precedes GFAP up regulation in diverse models of damage affecting different brain areas.

<p>Mice were administered the hippocampal neurotoxicant KA and mice and rats were administered the hippocampal neurotoxicant, TMT. Mice were killed by focused microwave irradiation to preserve steady-state phosphorylation of pSTAT3^{tyr 705} and phosphorylation was analyzed by quantification of scans of pSTAT3^tyr705 immunoblots. Representative immunoblot data from two different animals killed at each time point are presented above the quantitative data obtained from scans and are denoted by a bracket. Separate groups of mice were used to prepare total RNA from one side of the brain for qRT-PCR analysis of <i>Gfap</i> mRNA and to prepare total protein homogenates from the other side of the brain for ELISA of GFAP. Except for the representative immunoblot duplicates, all data points represent mean ± SEM, N = 5 (with the exception of pSTAT3 expression in TMT treated mice in which N = 2). Statistical significance was measured by one-way ANOVA with Fisher's LSD Method of post hoc analysis. Statistical significance of at least p<0.05 for the neurotoxicant exposed groups in comparison to saline controls is denoted by *. See Methods for dosing regimen.</p

Different neurotoxic insults damage neurons in different brain areas, leading to astrocyte and microglia activation.

<p>Activation of STAT3 (pTYR 705) represents a signaling event common to neurotoxic insults and neuroinflammation. Neurotoxicity-related damage results in astrogliosis dependent on activation of STAT3 but does not require upstream signaling from proinflammatory cytokines and chemokines. Suppression of this neuroinflammatory response to neurotoxicity does not affect expression of GFAP or pSTAT3. LPS causes brain-wide neuroinflammation (represented by flames) characterized by activation of microglia and elaboration of proinflammatory cytokines/chemokines but not neurodegeneration. Neuroinflammation-related activation of microglia due to LPS does not lead to astrogliosis but also is associated with activation of STAT3 suppressible by glucocorticoids. Neuroinflammation likely activates a separate STAT3 pathway, perhaps in microglia. Identification of upstream effectors in these STAT3 pathways will aid in defining and manipulating signal transduction events that likely play roles in repair and neuroimmune responses.</p

STAT3 activation is preceded by enhanced expression of proinflammatory ligands in multiple models of neurotoxicity.

<p>Mice were administered MPTP, AMP, METH, MDA, MDMA, KA and TMT with saline (0.9%) as a control and were killed at 6, 12 and 72 hours post dosing. Total RNA was prepared from striatum (MPTP, AMP, METH, MDA and MDMA) or hippocampus (KA and TMT) for qRT-PCR analysis of <i>Tnf-α</i>, <i>Osm</i>, <i>Ccl2</i> or <i>Lif</i>. All data points represent mean ± SEM, N = 5. Statistical significance was measured by one-way ANOVA with Fisher's LSD Method of post hoc analysis. Statistical significance of at least p<0.05 for the neurotoxicant exposed groups in comparison to saline controls is denoted by an asterisk. See Methods for dosing regimen (multi-dose METH).</p