This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms "siHBV") with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.

The different fate of liposomes among species has been discovered and mentioned in many studies, but the underlying mechanisms have not been explored. In the present work, we concentrated on the in vivo fate of PEGylated liposomes (sLip) in three commonly used species (mice, rats, and dogs). It was exhibited that the accelerated blood clearance (ABC) phenomenon and hypersensitivity in large animals (beagle dogs) was much more significant than that in rodents. We demonstrated that anti-PEG IgM (partially) and complement (mostly) determined the elimination of sLip and linked the distinct interspecies performances with the diverse complement capacity among species. Based on the data from animals and clinical patients, it was revealed that the fate of sLip in large animals was closer to that in humans, for the sufficient complement capacity could expose the potential adverse reactions caused by antiPEG antibodies. Our results suggested that the distinctive interspecies performances of sLip were highly related to the physiological variabilities among species, which should not be overlooked in the innovation and translation of nanomedicines.

Cancer patients suffer from the toxicity of chemotherapy. Antidote, given as a remedy limiting poison, is an effective way to counteract toxicity. However, few antidotes abrogate chemotoxicity without compromising the therapeutic efficacy. Herein, a rationally designed nanoantidote can neutralize chemo-agents in normal cells but not enter tumors and thus would not interfere with the efficacy of tumor treatment. The nanoantidote, consisting of a dendrimer core wrapped by reductive cysteine, captures Temozolomide (TMZ, the glioblastoma standard chemotherapy). Meanwhile, thanks to the blood-brain barrier (BBB) and the size of the nanoantidote, the nanoantidote cannot enter glioblastoma. In murine models, the nanoantidote distributes in normal tissues without crossing the BBB, so it markedly reduces the chemotoxicity of TMZ and retains the original TMZ therapeutic efficacy. With most nanotechnologies focusing on antitumor treatment, this detoxicating strategy demonstrates a nanoplatform to reduce chemotoxicity using physiology barriers and introduces a new approach to nanomedicine for cancer chemotherapy.

Glucose transporter (GLUT)-mediated transcytosis has been validated as an efficient method to cross the blood-brain barrier and enhance brain transport of nanomedicines. However, the transcytosis process remains elusive. Glycopeptide-modified nanodisks (Gly-A7R-NDs), which demonstrated high capacity of brain targeting via GLUT-mediated transcytosis in our previous reports, were utilized to better understand the whole transcytosis process. Gly-A7R-NDs internalized brain capillary endothelial cells mainly via GLUT-mediated/clathrin dependent endocytosis and macropinocytosis. The intracellular Gly-A7R-NDs remained intact, and the main excretion route of Gly-A7R-NDs was lysosomal exocytosis. Glycosylation of nanomedicine was crucial in GLUT-mediated transcytosis, while morphology did not affect the efficiency. This study highlights the pivotal roles of lysosomal exocytosis in the process of GLUT-mediated transcytosis, providing a new impetus to development of brain targeting drug delivery by accelerating lysosomal exocytosis.

Nanomedicines are of increasing scrutiny due to their improved efficacy and/or mitigated side effects. They can be integrated with many other therapeutics to further boost the clinical benefits. Among those, herbal medicines are arousing great interest to be combined with nanomedicines to exert synergistic effects in multifaceted mechanisms. The in vivo performance of nanomedicines which determines the therapeutic efficacy and safety is believed to be heavily influenced by the physio-pathological characters of the body. Activation of multiple immune factors, e.g., complement system, phagocytic cells, lymphocytes, and among many others, can affect the fate of nanomedicines in blood circulation, biodistribution, interaction with single cells and intracellular transport. Immunomodulatory effects and metabolic regulation by herbal medicines have been widely witnessed during the past decades, which alter the physio-pathological conditions and dramatically affect in vivo delivery of nanomedicines. In this review, we summarize recent progress of understanding on the in vivo delivery process of nanomedicines and analyze the major affecting factors that regulate the interaction of nanomedicines with organisms. We discuss the immunomodulatory roles and metabolic regulation by herbal medicines and their effects on in vivo delivery process of nanomedicines, as well as the prospective clinical benefits from the combination of nanomedicines and herbal medicines.

Nanovaccines are of increasing scrutiny due to their plasticity in size, composition, and surface properties to enhance antigenicity. However, inevitable absorption of plasma proteins affects the in vivo fate of nanovaccines by reshaping biological identity. Herein IgM was validated as a self-adjuvant by regulating antigen-presenting cells recognition of liposome-based nanovaccines. DCDX-modified liposomes with loading of ovalbumin (DCDX-sLip/OVA) heavily absorbed IgM via electrostatic interaction, demonstrating significant splenic B cells targeting. IgM absorbed on DCDX-sLip/OVA enhanced antigen uptake and presentation by both IgM-complement and IgM-FcμR pathways. DCDX-sLip/OVA induced a stronger IgG1 titer than ovalbumin-loaded plain liposomes (sLip/OVA) while maintaining a comparably high level of IgG2a titer with high biosafety, indicating that IgM absorption after DCDX modification could improve the antigenicity by enhancing the Th2-polarized immune response. The present work suggested manipulation of IgM absorption may provide a new impetus to improve in vivo performance of nanovaccines.

Mice are arguably the most important tool in the preclinical evaluation of liposomes; however, the effects of inter-strain physiological variabilities on in vivo performance of liposomes have been seriously overlooked. The present study validated that plasma proteins (PPs) and the capability of mononuclear phagocyte system (MPS) (typically expressed by phagocytosis rate, K) were mice strain-dependent. Physiological variabilities in PPs and the phagocytosis rate jointly contributed to the inter-strain inconsistency of pharmacokinetic (PK) profiles of liposomes. For the PPs sensitive liposomes (such as plain PEGylated liposomes and folic acid functionalized PEGylated liposomes), inter-strain variabilities in PK profiles could be calibrated using the corrected phagocytic rate (KC = K×(c × Ig)/(alb×apo)), where c, Ig, alb and apo were respective the total content of complement proteins, immunoglobulins, albumin and apolipoproteins. While for the PPs insensitive liposomes (e.g., cRGD functionalized liposomes), phagocytic rate could be directly used to calibrate inter-strain difference of liposome PK profiles. Our data also warn that the reciprocal interaction between payloads and organisms would be much more complicated than that between liposomes and organisms, thus independent investigation should be conducted for each individual therapeutic agent.

Prevalent deposition of plasma proteins on nano-surface alters the synthetic identity of liposomes in blood circulation. The key plasma protein(s) that can dominate in vivo fate of liposomes are of central importance for preclinical design and precise medication of liposome-based therapeutics. Herein, natural IgM, but not IgG, is identified to ubiquitously absorb on liposomal surface and takes the lead in complement activation of different species. The absorbed natural IgM, which negatively correlates with the in vivo performance of liposomes, becomes a potential indicator to guide the de novo design and optimization of liposomes. More importantly, the varying natural IgM levels in cancer patients may be one of the causal factors for clinical differences in response to liposome-based therapeutics. Clinical monitoring of the natural IgM level and its binding with liposomes becomes crucial to optimize the therapeutic regimen prior to the application of liposome-based therapeutics.

Folic acid (FA) has been extensively exploited to facilitate targeted delivery of nanomedicines by recognizing the folate receptor-α (FR-α) overexpressed in many human cancers. Unfortunately, none have been approved for clinical use yet. Here we reveal that FA functionalization induces heavy natural IgM absorption on the liposomal surface, depriving FA of receptor recognition and accelerating complement activation in vivo. FA functionalization does not enhance distribution of liposomes in FR-α-overexpressed tumors in comparison to plain liposomes (without FA), but leads to aggravated capture of liposomes by macrophages in the tumor, liver, and spleen. In addition, FA-functionalized polymeric nanoparticles are also vulnerable to natural IgM absorption. This work highlights the pivotal roles of natural IgM in regulating in vivo delivery of FA-functionalized nanomedicines. Due to the prevalent association of immune disorders and varying levels of immunoglobulins with cancer patients, extraordinary cautiousness is urged for clinical translation of FA-enabled targeted delivery systems.

Cholera toxin subunit B (CTB) is the nontoxic moiety of cholera toxin. It can target the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasculature, and glioblastoma cells. Thus, CTB has been utilized as a multifunctional molecule for targeted therapy of glioblastoma. Here, we describe a detailed method for preparation of CTB functionalized paclitaxel (PTX)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles. This unique modification can guide nanoparticles across the BBB and target glioblastoma cells. The characterization of nanoparticles such as size, zeta potential, morphology, drug loading, and encapsulation efficiency is shown in this chapter.

Targeted drug delivery is emerging as a promising strategy to achieve better clinical outcomes. Actively targeted drug delivery that utilizes overexpressed receptors or antigens on diseased tissues is receiving increasing scrutiny, especially due to the uncertainty of existence of the enhanced permeability and retention (EPR) effect in cancer patients. Peptide ligands are advantageous over other classes of targeting ligands due to their accessibility of high-throughput screening, ease of synthesis, high specificity and affinity, etc. In this review, we briefly summarize the resources of peptide ligands and discuss the pitfalls and perspectives of peptide ligand-mediated targeted delivery of nanomedicines.

Protein corona presents a major obstacle to bench-to-bedside translation of targeted drug delivery systems, severely affecting targeting yields and directing unfavorable biodistribution. Corona-mediated targeting provides a new impetus for specific drug delivery by precisely manipulating interaction modes of functional plasma proteins on nano-surface. Here bio-inspired liposomes (SP-sLip) were developed by modifying liposomal surface with a short nontoxic peptide derived from Aβ 1-42 that specifically interacts with the lipid-binding domain of exchangeable apolipoproteins. SP-sLip absorb plasma apolipoproteins A1, E and J, consequently exposing receptor-binding domain of apolipoproteins to achieve brain-targeted delivery. Doxorubicin loaded SP-sLip (SP-sLip/DOX) show significant enhancement of brain distribution and anti-brain cancer effect in comparison to doxorubicin loaded plain liposomes. SP-sLip preserve functions of the absorbed human plasma ApoE, and the corona-mediated targeting strategy works in SP modified PLGA nanoparticles. The present study may pave a new avenue to facilitate clinical translation of targeted drug delivery systems.

Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Officinalis, demonstrating high potency in promoting nonrapid eye movement (NREM) sleep by modulating the benzodiazepine site of the GABAA receptor. However, the clinical use of HNK in the treatment of insomnia is restricted by its extremely low oral bioavailability. In the present work, enhanced oral bioavailability of HNK was achieved by loading it into poly lactide-glycolide acid microparticles (HNK-MP). After oral administration, HNK-MP demonstrated 15-fold increase of AUC0-12 h in comparison to free HNK. The maximum blood concentration ( Cmax) of HNK in HNK-MP-treated rats was 3.6 μg/mL at 2 h after oral administration, which was 6.5-fold of that in free HNK-treated rats. Oral administration of HNK-MP (20 mg/kg) efficiently increased NREM sleep by 60% by enhancing the transition from wakefulness to NREM sleep in rats. The biosafety of HNK-MP was assessed in vivo, and no damage occurred in the gastrointestinal tract. The present study provides a promising oral HNK formulation for the treatment of insomnia.

Targeted drug delivery is emerging as a promising strategy to achieve better clinical outcomes. Actively targeted drug delivery that utilizes overexpressed receptors or antigens on diseased tissues is receiving increasing scrutiny, especially due to the uncertainty of existence of the enhanced permeability and retention (EPR) effect in cancer patients. Peptide ligands are advantageous over other classes of targeting ligands due to their accessibility of high-throughput screening, ease of synthesis, high specificity and affinity, etc. In this review, we briefly summarize the resources of peptide ligands and discuss the pitfalls and perspectives of peptide ligand-mediated targeted delivery of nanomedicines.

脂质体是在临床阶段应用最为广泛的纳米药物载体,进入血循环后迅速吸附血浆蛋白,其表面形成的蛋白冠可直接影响脂质体体内各项性能.深入研究脂质体自身性质、血浆蛋白组分与脂质体体内性能间相互关系,是加速新型脂质体药物临床开发和指导脂质体药物临床合理用药的必经之路.本文分别从影响脂质体蛋白冠的因素、蛋白冠对脂质体体内性能的影响和基于蛋白冠的脂质体药物设计三个方面进行综述,为脂质体药物相关研究提供参考.

The side effects of docetaxel have limited its antitumor performances in the treatment of nonsmall cell lung cancer (NSCLC). To address the problem, baicalein, a bioactive flavone that exhibits antitumor activity, was combined with docetaxel so as to achieve better efficacy and lower toxicity. The combination treatment enhanced the stabilization of microtubules and halted the cell‐cycle progression, thus synergistically inhibiting the proliferation and inducing the apoptosis of A549 cells and Lewis lung carcinoma cells. The decreased expression of Cyclin‐dependent kinase 6 and Cyclin B1 confirmed its regulation in cell cycle, with β‐catenin being an important upstream effector, as evidenced by the decreased expression in the cytoplasm and nucleus as well as the attenuated aggregation in the nucleus. Furthermore, baicalein plus docetaxel evinced better antitumor efficacy by the suppressed tumor growth, increased apoptosis, and decreased tumor angiogenesis in vivo, with no increased toxicity discovered in both tumor‐bearing and non‐tumor‐bearing mice, and an improvement in therapeutic index. This study has demonstrated that baicalein plus docetaxel is an appropriate combination simultaneously with augmented antitumor efficacy and acceptable safety, which might be a promising strategy for patients with advanced NSCLC.

Targeting ligands are anticipated to facilitate the precise delivery of therapeutic agents to diseased tissues; however, they may also severely affect the interaction of nanocarriers with plasma proteins. Here, we study the immunocompatibility of brain-targeted liposomes, which inversely correlates with absorbed natural IgM. Modification of long, stable positively charged peptide ligands on liposomes is inclined to absorb natural IgM, leading to rapid clearance and enhanced immunogenicity. Small peptidomimetic D8 developed by computer-aided peptide design exhibits improved immunocompatibility by attenuating natural IgM absorption. The present study highlights the effects of peptide ligands on the formed protein corona and in vivo fate of liposomes. Stable positively charged peptide ligands play double-edged roles in targeted delivery, preserving in vivo bioactivities for binding receptors and long-term unfavorable interactions with the innate immune system. The development of D8 provides insights into how to rationally design immunocompatible drug delivery systems by modulating the protein corona composition.

胶质瘤是中枢神经系统中最常见的恶性肿瘤,全基因组分子图谱已揭示不同类型胶质瘤的遗传学和表观分子生物学特征.2016年世界卫生组织在经典的组织病理学诊断基础上,将分子标志物纳入胶质瘤的分类中,为胶质瘤的诊断提供了一套新的金标准,有望对临床上胶质瘤的干预和预后产生重要意义.

Glioma is among the most formidable brain cancers due to location in the brain. Cholera toxin subunit B (CTB) is investigated to facilitate multifunctional glioma-targeted drug delivery by targeting the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasulature, and glioma cells. When modified on the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CTB-NPs), CTB fully retains its bioactivity after 24 h incubation in the fresh mouse plasma. The formed protein corona (PC) of CTB-NP and plain PLGA nanoparticles (NP) after incubation in plasma is analyzed using liquid chromatography tandem massspectrometry (nano-LC-MS/MS). CTB modification does not alter the protein components of the formed PC, macrophage phagocytosis, or pharmacokinetic profiles. CTB-NP can efficiently penetrate the in vitro BBB model and target glioma cells and human umbilical vascular endothelial cells. Paclitaxel is loaded in NP (NP/PTX) and CTB-NP (CTB-NP/PTX), and their antiglioma effects are assessed in nude mice bearing intracranial glioma. CTB-NP/PTX can efficiently induce apoptosis of intracranial glioma cells and ablate neovasulature in vivo, resulting in significant prolongation of survival of nude mice bearing intracranial glioma (34 d) in comparison to those treated with NP/PTX (29 d), Taxol (24 d), and saline (21 d). The present study suggests a potential multifunctional glioma-targeted drug delivery system enabled by cholera toxin subunit B.