Citation: Ganbold, T.; Bao, Q.; Xiao, H.; Zurgaanjin, D.; Liu, C.; Han, S.; Hasi, A.; Baigude, H. Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice. Nanomaterials 2022, 12, 2072. https://doi.org/10.3390/ nano12122072 Academic Editor: Angelina Angelova Received: 22 May 2022 Accepted: 14 June 2022 Published: 16 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nanomaterials Article Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice Tsogzolmaa Ganbold 1,2 , Qingming Bao 1 , Hai Xiao 1 , Dolgorsuren Zurgaanjin 1 , Caifeng Liu 1 , Shuqin Han 1 , Agula Hasi 2, * and Huricha Baigude 1, * 1 Inner Mongolia Key Laboratory of Mongolian Medicinal Chemistry, School of Chemistry & Chemical Engineering, Inner Mongolia University, Hohhot 010020, China; tsogi001@yahoo.com (T.G.); qingming_@mail.imu.edu.cn (Q.B.); haixiao@mail.imu.edu.cn (H.X.); dolgorsuren@mail.imu.edu.cn (D.Z.); liucaifenga@yeah.net (C.L.); chem-hshq@imu.edu.cn (S.H.) 2 School of Life Sciences, Inner Mongolia University, Hohhot 010020, China * Correspondence: hasind@sina.com (A.H.); hbaigude@imu.edu.cn (H.B.) Abstract: Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflam- matory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroin- flammation 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. Keywords: lipid nanoparticles; siRNA delivery; microglia; neuroinflammation; stroke 1. Introduction Neuroinflammation is the major cause of secondary brain injury after stroke [1]. Microglia, the resistant immune cells in central nervous systems (CNS), are the first defense line after brain injury. In response to cerebral ischemia, microglia activation appears to be a double-edged sword, which can be categorized into two opposite types: neuroprotective M2 phenotype and neurotoxic M1 phenotype [2]. M1-phenotype mi- croglia release multiple proinflammatory factors, including TNFα, IL-1β and reactive oxygen species (ROS), via induction of a transcriptional reprogramming of genes that promote posttranscriptional activation of inflammatory signaling pathways that control the stability of nuclear factor-κB (NF-κB) and hypoxia-induced factor 1 (HIF-1) to exacer- bate secondary brain injury [3], while those of M2 phenotype release anti-inflammatory factors such as IL-10, TGF-β and IL-4 to promote brain recovery after ischemic stroke [4]. In brain hypoxia during stroke, sustained neuroinflammation by overactivated M1 mi- croglia damages neuron and cerebrovascular endothelial cells, which induces tissue destruction and worsens functional outcome [5]. Therefore, microglia are thought to be a key target for effective therapeutic intervention against ischemic stroke by regulating M1/M2 phenotypes after ischemic stroke [6]. Nanomaterials 2022, 12, 2072. https://doi.org/10.3390/nano12122072 https://www.mdpi.com/journal/nanomaterials