Synthesis of Lipidated Ligands and Formulation of Glia-Specific LNPs for RNAi-Mediated BBB Protection.

Pro-inflammatory polarization of microglia and astrocytes results in neuroinflammation and blood-brain barrier (BBB) disruption after a primary traumatic brain injury (TBI). Herein, we demonstrate that the dual-ligand functionalized lipid nanoparticles (AM31 LNPs) were actively and specifically internalized by microglia and astrocytes via mannose receptor (MR)- and adenosine receptor (AR)-mediated endocytosis, respectively, in a mouse model of TBI. Systemic administration of AM31 LNPs carrying siRNA against p65 resulted in internalization by the glial cells in the peri-infarct region and a robust knockdown of p65 at both mRNA and protein levels in these cells, leading to significant down-regulation of key pro-inflammatory cytokines and up-regulation of key anti-inflammatory cytokines. AM31 LNP-mediated silencing of p65 ameliorated TBI-induced BBB disruption. Our data proved that AM 31 LNP is a promising vehicle for RNA therapeutics for targeting microglia and astrocytes in neural disorder.

Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

Tumor targeted siRNA delivery by adenosine receptor-specific curdlan nanoparticles.

Aminated curdlan derivatives are highly effective nucleic acid carriers. Previously, we proved that the ligand-functionalized curdlan derivatives have greatly enhanced cell type specificity induced by receptor-mediated internalization in vitro. In this study, to improve biocompatibility and enhance tumor-targeting efficacy of the curdlan derivative, we pegylated the adenosine functionalized amino curdlan derivative (denoted by pAVC polymer). We confirmed that the uptake of pAVC polymer carrying siRNA by tumor cells was adenosine receptor (AR)-dependent and was specifically inhibited by AMP but not by GMP. The pAVC polymers not only preserved the receptor recognition and exhibited significantly decreased cytotoxicity but also showed remarkable tumor targeting efficiency in vivo. The nanoparticles formulated from siRNA (against STAT3) and pAVC4 polymer, which bears the highest degree of PEG substitution, delivered siRNA highly specifically to tumor tissue, knocked down STAT3, and inhibited tumor growth. The pAVC polymers may be a promising carrier for tumor specific delivery of nucleic acid drugs.

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen-glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.

Synthesis of PEGylated cationic curdlan derivatives with enhanced biocompatibility.

Cationic polysaccharides have shown excellent ability of nucleic acids delivery. However, cationic curdlan derivatives with high degree of amination cause damage to the cell membrane and induce considerable cytotoxicity, limiting their in vivo application. Herein, we synthesized PEGylated 6-amino-6-deoxy-curdlan derivatives containing cleavable disulfide bonds. The resulting polymers (denote 6AC-2S PEGx) not only showed high affinity to siRNA but also exhibited significantly decreased cytotoxicity and hemolysis effect, while showing remarkable in vitro transfection efficiency. In vivo study demonstrated that 6AC-2S PEG40, which had a lower LD50 value than that of 6AC-100, did not cause liver damage, as the i.v. injection of 6AC-2S PEG40 to mouse did not increase serum level of ALT/AST. Furthermore, tissue distribution results showed that 6AC-2S PEG40 successfully delivered siRNA to liver, lung and spleen. Collectively, our data confirmed that PEGylation can increase the biocompatibility of cationic curdlan derivatives, which is a promising carrier for nucleic acid therapeutics.

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor.

Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A2B receptor. The internalization of siRNA loaded CuAMP4 by cancer cells was inhibited by free AMP as well as endocytosis inhibitors. Moreover, knockdown of A2BR by siRNA, or pre-treatment of the cells with anti-A2BR antibody, strongly inhibited the cellular uptake of CuAMP4. Our findings confirmed that A2BR can be utilized for cell type specific siRNA delivery, and CuAMP4 NP may be a promising delivery system for cancer cell targeted delivery of therapeutic siRNAs.

Modulation of Microglia Polarization through Silencing of NF-κB p65 by Functionalized Curdlan Nanoparticle-Mediated RNAi.

Microglia polarization plays an important role in poststroke recovery. Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory (M2) polarization of microglia are potential therapeutic strategies for inflammation reduction and neuronal recovery after stroke. Here, we evaluated the central nervous system (CNS)-targeted short interfering RNA (siRNA) delivery ability of functionalized curdlan nanoparticles (CMI) and investigated the nuclear factor-κB (NF-κB) p65 silencing efficiency of CMI-mediated siRNA in microglia, as well as the resulting neuroprotective effect of microglia polarization and neuroprotection in vitro and in vivo. The systemic delivery of NF-κB p65 siRNA (sip65) complexed to CMI nanoparticles in the mouse model of transient middle cerebral artery occlusion (tMCAO) resulted in the distribution of siRNA in microglia and significant silencing in NF-κB p65 in the peri-infarct region. Knockdown of NF-κB p65 resulted in M1 to M2 phenotypic transition of microglia, evidenced by the change in the expression pattern of signature cytokines as well as inducible nitric oxide synthase and CD206. Moreover, the CMI-mediated silencing of p65 increased the density of neurons and decreased pyknosis and edema in the peri-infarct region. Assessment of the neurological deficit score on the Bederson scale revealed a significantly reduced score in the mouse model of tMCAO treated with the sip65/CMI complex. Collectively, our data suggest that CMI nanoparticles are a promising CNS-targeting siRNA delivery system, and NF-κB p65 may be a potential therapeutic target for inflammation reduction and poststroke recovery.

Bioactive components of ethnomedicine Eerdun Wurile regulate the transcription of pro-inflammatory cytokines in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Mongolian medicine Eerdun Wurile (EW) has remarkable neural recovery effect, and has been playing a key role in the clinical treatment of neurological disorders including ischemic stroke in Inner Mongolia Autonomous Region of China. The preliminary pharmacological studies in animal suggested that EW regulates the expression of trophic factors in brain lesion and may also balance the polarization of activated microglia (Gaowa et al., 2018). AIM OF THE STUDY: The pool of leading bioactive chemicals underlying the therapeutic effects of EW has not been identified. Therefore, the mechanism of action of EW is poorly understood. This study was aimed to identify the major group of compounds that contribute to the inhibition of neuroinflammation during stroke recovery through regulation of microglia polarization. MATERIALS AND METHODS: The extracts of EW in different solvents were evaluated for their inhibitory ability of cytokine (IP-10) expression in LPS stimulated BV2 cells. The most effective extract (of petroleum ether extract) was further separated to 18 fractionations on a semi-preparative HPLC column, which were assess for the IP-10 down-regulation efficiency by RT-qPCR. The potent isolate was further fractionated in 12 fractions, which showed fewer peaks. The fraction 6 from this isolates, which remarkably down-regulates cytokines expression including IP-10, TNFα and IL-1ß, was analyzed on UPLC-qTOF MS. The key chemicals were measured for their cytokine inhibition in BV2 cells and mouse primary microglia. RESULTS: After two consecutive fractionating by preparative HPLC, petroleum ether extraction of EW gave 12 fractions with relatively distinctive chromatograms. A particular fraction (fraction 6) preserved the inhibitory effects on expression of pro-inflammatory cytokines including IP-10, TNFα, IL-1ß and iNOS. The result of UPLC-qTOF MS analysis showed that the fraction contains 21 chemicals including costunolide, alantolactone, myristicin and linolenic acid, which significantly down-regulate the expression of key pro-inflammatory cytokines in LPS stimulated BV2 cells as well as mouse primary microglia. CONCLUSION: Collectively our data suggest that the bioactive chemical pool which is responsible for the therapeutic effects of EW can be extracted in petroleum ether, and fractionated to a relatively small multiple components. Such components include known anti-inflammatory chemicals, which may contribute to the possible microglia polarization in brain lesion during the recovery of ischemic stroke.

Sugar Functionalized Synergistic Dendrimers for Biocompatible Delivery of Nucleic Acid Therapeutics.

Sugars containing cationic polymers are potential carriers for in vitro and in vivo nucleic acid delivery. Monosaccharides such as glucose and galactose have been chemically conjugated to various materials of synergistic poly-lysine dendrimer systems for efficient and biocompatible delivery of short interfering RNA (siRNA). The synergistic dendrimers, which contain lipid conjugated glucose terminalized lysine dendrimers, have significantly lower adverse impact on cells while maintaining efficient cellular entry. Moreover, the synergistic dendrimers complexed to siRNA induced RNA interference (RNAi) in the cells and profoundly knocked down green fluorescence protein (GFP) as well as the endogenously expressing disease related gene Plk1. The new synergic dendrimers may be promising system for biocompatible and efficient siRNA delivery.