Universal and Translational Nanoparticulate CpG Adjuvant

CpG, an agonist of toll-like receptor 9 (TLR9), has become a novel adjuvant that substantially potentiates cellular immunity. However, this agonist may increase systemic toxicity by diffusing into blood after administration and is difficult to be internalized by immune cells to reach TLR9 located in endosomes as a result of the characteristics of negative charge of CpG. Here, we applied a scalable and controllable flash nanocomplexation technology to prepare nanoparticulate CpG adjuvant (npCpG), CpG encapsulated in a physical cross-linking network of protamine and TPP. The nanoadjuvant could redirect CpG into draining lymph nodes to reduce systemic diffusion to improve safety. Further, a combination of npCpG and influenza H1N1 hemagglutinin antigen showed excellent humoral and cellular immunity, evoking high levels of antibodies and cytokines and inducing a great expansion of splenocytes in immunized mice. Also, the nanoadjuvant combined with ovalbumin antigen led to a potent cytotoxic T-cell response, substantially inhibited tumor growth, and improved the survival rate of mice in a melanoma model. This study showed the universal performances of npCpG in infectious disease prevention and tumor immunotherapy to demonstrate the translational potential

Salt Cocrystal and Salt of Marbofloxacin with Butenedioic Acid: Impact of <i>cis</i>–<i><i>trans</i></i> Isomerism of Coformer on the Conformation and Properties of Marbofloxacin

Based on the study of the effect of positional isomerism of coformer functional groups on the cocrystallization and physicochemical properties of the active pharmaceutical ingredients, the impact of cis–trans isomeric butenedioic acid as coformers on the conformation, crystal structure, and its physicochemical properties of marbofloxacin was further explored. In this work, fumaric acid (FA) and maleic acid (MA) with different configurations were chosen as coformers to synthesize the pharmaceutical salt cocrystal (MBF-FA-H2FA) and salt (MBF-MA) of marbofloxacin (MBF), and their structures were fully characterized. Significant differences between the conformations of marbofloxacin in the salt cocrystal and in salt were found. In the salt cocrystal, the N atom of the piperazine group from marbofloxacin is coplanar with the pyridone ring, and the whole is straight like fumaric acid, whereas the marbofloxacin piperazine group in the salt is bent like the maleic acid configuration. Furthermore, the conformational variability of marbofloxacin in the salt cocrystal and the salt resulted in different crystal structures and opposite physicochemical properties. Notably, both multicomponent crystals have a surface hydrophilic intercalation structure. However, the salt cocrystal and salt exhibited different solubility and permeability. Specifically, the MBF-MA salt showed improved solubility and permeability, while the MBF-FA-H2FA salt cocrystal showed a decreased solubility and permeation rate compared to MBF. In addition, in vitro bacterial inhibitory activity assays indicated that the MBF-MA salt has stronger inhibitory activity against Gram-negative and Gram-positive bacterial strains than the MBF-FA-H2FA salt cocrystal and pure MBF

Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age

<div><p>Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFβ1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.</p></div

Well-Defined Poly(α-amino-δ-valerolactone) via Living Ring-Opening Polymerization

This article demonstrates the synthesis of a new kind of cationic poly­(δ-valerolactone) with primary amino groups at α-positions (poly­(α-NH₂-VL)) via ring-opening polymerization (ROP) of α-NHBoc-valerolactone (α-NHB-VL) followed by a simple deprotection reaction. The ROP of α-NHB-VL using benzyl alcohol as an initiator and DBU/TU (1,8-diazabicyclo[5.4.0]­undec-7-ene/thiourea) as a catalytic system in THF at room temperature afforded poly­(α-NHB-VL) with narrow molecular weight distribution. The ¹H NMR and MALDI-TOF MS analysis of poly­(α-NHB-VL) indicated that each polymeric chain was capped by the initiator. Kinetic experiments confirmed the living nature of the DBU/TU-catalyzed ROP of α-NHB-VL in THF. The copolymerization result indicated that the polymerization activity of α-NHB-VL is comparable to that of ε-caprolactone (CL) and VL. In addition, block copolymers containing poly­(α-NHB-VL) were successfully synthesized regardless of whether hydrophilic PEG or hydrophobic PCL was used as the macroinitiator. Moreover, water-soluble poly­(α-NH₂-VL) was obtained by treatment with trifluoroacetic acid (TFA). It was found that poly­(α-NH₂-VL) degraded more slowly at pH 5.5 than at pH 7.4 through a hydrolysis kinetics study

Iba1 immunofluorescence images of the ipsilateral dorsal horns from naïve young (YN) and middle-aged (MN) rats rendered in 3D.

<p>Upper Panels: individual optical sections (1.25 μm intervals through 25 μm thick sections) were acquired with a LSCM and 40x 1.4 NA objective lens and then recombined and rendered as three-dimensional images. Lower Panels: Two-photon immunofluorescence 3D renderings of Iba1 positive dorsal horn microglia from young and middle-aged naïve (YN and MN). Image stacks (0.44 μm optical sections through 25 μm thick specimens) were obtained using a two-photon laser scanning confocal microscope and 100 X 1.4 NA objective lens. Each stack set was recombined to create the 3D rendering. A single plane of the 3D image is shown for each. Scale bar = 25 μm. Rotatable 3D images are also available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.s004" target="_blank">S2</a> Files.</p

Lumbar Spinal Cord Gene Expression Comparing Sham and CCI: Effects of Age.

<p>LSC ipsilateral hemisections were removed on day 7. Expression levels in lumbar spinal cord were normalized to the geomean of Hprt1 and Pplb expression and the ratios multiplied by 100 and presented as the mean ratios +/- SD. Two-way ANOVA results are shown with contrasts and associated p values. Numbers of animals (n) assayed per group are shown. Bolded/italicized genes indicate specific contrast differences p < 0.05</p><p>* indicates difference trending to significance. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.t002" target="_blank">Table 2</a>.</p><p>Lumbar Spinal Cord Gene Expression Comparing Sham and CCI: Effects of Age.</p

Neuroimmune gene expression profile in lumbar spinal cords of naïve young and middle-aged rats.

<p>Expression levels were normalized to the geomean of Hprt1 and Pplb expression and the ratios multiplied by 100. Results are presented as means +/- SD. Significance of differences between YN v MN * p < 0.0015.</p

Gene Expression Profile of Middle-Aged Sham and CCI DRG at Day 7.

<p>Two-way ANOVA results (normalized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.t001" target="_blank">Table 1</a>) of dorsal root ganglia expression levels are shown with contrasts and associated p values and numbers of animals assayed per group. Bolded/italicized genes indicate specific contrast differences p < 0.05</p><p>* indicates difference trending to significance.</p><p>Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134394#pone.0134394.t002" target="_blank">Table 2</a>.</p><p>Gene Expression Profile of Middle-Aged Sham and CCI DRG at Day 7.</p

Gene Expression Profile of Young Sham and CCI DRG at Day 7.

<p>Gene Expression Profile of Young Sham and CCI DRG at Day 7.</p

Representative Iba1 immunofluorescence images of young and middle aged of lumbar spinal cord dorsal horns from post-CCI day 7 animals, ipsilateral or contralateral to injury.

<p>Immunofluorescence confocal images of the dorsal horns stained with Iba1 antibody were combined with corresponding transmitted light images. Young (YCCI) and middle-aged (MCCI) dorsal horns ipsilateral (IPSL) to injury are compared to the contralateral (CL) sides. Scale bars = 100 μm.</p