Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

Enhancing anti-tumor therapy with agmatine-cholesterol conjugate liposomes: in vitro and in vivo evidence.

In this study, we synthesized a novel compound, agmatine-cholesterol conjugate (AG-Chol), to enhance the anti-tumor activity of drug-loaded liposomes. We replaced cholesterol with AG-Chol in preparing doxorubicin hydrochloride (DOX) liposomes by using an active loading method for DOX. We assessed the physical and chemical properties of the resulting AG-Liposomes and evaluated their efficacy in vitro and in vivo. The results showed that AG-Liposomes were stable with high encapsulation efficiency. Compared with the control liposomes, AG-Liposomes exhibited a slower drug release rate in the release medium at pH 6.8. The in vitro cell experiments demonstrated that AG-Liposomes had higher tumor cell uptake rate, stronger migration inhibition rate, higher apoptosis rate, better anti-clonogenic ability, and higher lysosome escape ability than the control liposomes. In vivo distribution results demonstrate that liposomes prepared with AG-Chol instead of cholesterol can significantly enhance their tumor targeting abilities and reduce their distribution to non-targeted sites. In vivo tumor suppression experiments showed that AG-Liposomes had a higher tumor suppression rate than the control liposomes without causing apparent toxicity to normal tissues, as evidenced by histological staining. Therefore, substituting cholesterol with AG-Chol in the preparation of liposomes can result in enhanced lysosome escape, improved tumor targeting, and increased efficacy of anti-tumor drugs.

Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules is one of the most promising approaches for delivering siRNA in vivo. The rate of molecular weight-dependent siRNA renal clearance is critical for the efficiency of this process. In this study, we prepared cholesterol-containing supramolecular complexes containing from three to eight antisense strands and examined their accumulation and silencing activity in vitro and in vivo. We have shown for the first time that such complexes with 2'F, 2'OMe, and LNA modifications exhibit interfering activity both in carrier-mediated and carrier-free modes. Silencing data from a xenograft tumor model show that 4 days after intravenous injection of cholesterol-containing monomers and supramolecular trimers, the levels of MDR1 mRNA in the tumor decreased by 85% and 68%, respectively. The in vivo accumulation data demonstrated that the formation of supramolecular structures with three or four antisense strands enhanced their accumulation in the liver. After addition of two PS modifications at the ends of antisense strands, 47% and 67% reductions of Ttr mRNA levels in the liver tissue were detected 7 days after administration of monomers and supramolecular trimers, respectively. Thus, we have obtained a new type of RNAi inducer that is convenient for synthesis and provides opportunities for modifications.

Optimization design of sialic acid derivatives enhances the performance of liposomes for modulating immunosuppressive tumor microenvironments.

AIMS: Sialic acid derivatives (SA-derivatives) provide a nanomedicine platform for tumor-targeted delivery and treatment, and allow modulation of immunosuppressive tumor microenvironments with excellent therapeutic effects. Further, the multi-reactive groups of sialic acid (SA) contribute to the diversity of SA derivatives, which inevitably has implications for drug delivery systems and tumor therapy. However, relevant research remains lacking at present. Therefore, this study aimed to explore the effects of SA derivatives on SA-mediated drug delivery systems. MAIN METHODS: Four SA-derivatives with different linking bonds (ester and amide bonds), different linking groups (hydroxyl and carboxyl), and different linking objects (cholesterol, octadecanoic acid, and octadecylamine) were synthesized and the respective SA derivative-modified doxorubicin liposomes were prepared. In-depth research was conducted using both cells and animals. KEY FINDINGS: We found that an SA-cholesterol conjugate (SA-CH; linking bond, amide bond; linking group, carboxyl; linking object, cholesterol) could improve liposome stability, reduce liposome adsorption to plasma proteins, and enhance the targeting of liposomes for killing tumor-associated macrophages (TAMs). Reduced TAMs in the immunosuppressive tumor microenvironment lead to enhanced tumor infiltration of CD8+ T cells. SIGNIFICANCE: The results of this experiment provide clarity for research and development on SA-derivatives and a theoretical basis for clinical trials of SA-derivative-modified nanoparticles.

Pheophorbide A and SN38 conjugated hyaluronan nanoparticles for photodynamic- and cascadic chemotherapy of cancer stem-like ovarian cancer.

In this study, we designed photo-triggered reactive oxygen species (ROS)-generating pheophorbide A and ROS-cleavable thioketal-SN38 conjugated hyaluronan-cholesterol nanoparticles (PheoA-SN38-HC NPs). And we observed the combined therapeutic effects of PheoA-SN38-HC NPs against HEY-T30 human ovarian cancer (OC) model. Clinical Proteomic Tumor Analysis Consortium (CPTAC) data showed that the expression of cancer stem cell (CSC) markers (CD44, ALDH1A1, and CD117) is highly associated with poor clinical outcomes in OC patients. We proved that HEY-T30 cells overexpress CSC markers and much more invasive than other cancer cells. Flow cytometry (FACS) and microscopic analysis revealed the active targeting property of PheoA-SN38-HC NPs to CD44+ HEY-T30 cells. Moreover, the combination therapeutic effect of PheoA-SN38-HC NPs was clearly demonstrated against in vitro HEY-T30 cells and an in vivo xenograft mouse model. In particular, the paracrine cytotoxic effect of SN38 probably compensates the locoregional therapeutic limitation of photodynamic therapy.

Evaluation of cell surface reactive immuno-adjuvant in combination with immunogenic cell death inducing drug for in situ chemo-immunotherapy.

Apoptotic cells and cell fragments, especially those produced as a result of immunogenic cell death (ICD), are known to be a potential source of cancer vaccine immunogen. However, due to variation between tumours and between individuals, methods to generate such preparations may require extensive ex vivo personalisation. To address this, we have utilised the concept of in situ vaccination whereby an ICD inducing drug is injected locally to generate immunogenic apoptotic fragments/cells. These fragments are then adjuvanted by a co-administered cell reactive CpG adjuvant. We first evaluate means of labelling tumour cells with CpG adjuvant, we then go on to demonstrate in vitro that labelling is preserved following apoptosis and, furthermore, that the apoptotic body-adjuvant complexes are readily transferred to macrophages. In in vivo studies we observe synergistic tumour growth delays and elevated levels of CD4+ and CD8+ cells in tumours receiving adjuvant drug combination. CD4+/CD8+ cells are likewise elevated in the tumour draining lymph node and activated to a greater extent than individual treatments. This study represents the first steps toward the evaluation of rationally formulated drug-adjuvant combinations for in situ chemo-immunotherapy.

Insight into the siRNA transmembrane delivery-From cholesterol conjugating to tagging.

Small interfering RNA (siRNA), combining the features of unprecedented potency, target-specificity, and the unique sequence-based disease-intervention model, has received immense considerations over the past decades in the academia and pharmaceutical industry. siRNA fits the criteria of being drug-likely enough to meet with the therapeutic purpose, but its clinical translation has been impeded for a long time by the poor efficiency of in vivo delivery. To reach the cytosol where the RNA interference (RNAi) takes place, siRNA delivery faces a serial of systemic and cellular barriers, especially the endosomal sequestration that would prevent the majority of siRNA from cytosol entry. Transmembrane delivery of siRNA represents a new avenue for efficient delivery by bypassing the endosomal pathway. This rationale is bolstered by the high efficiency of viral entry by membrane fusion, but rarely pursued by artificial siRNA delivery systems. Here, this article provides an opinion of transmembrane delivery by hydrophobic modulation of siRNA. We give a brief introduction of the current siRNA delivery modes, including the hydrophobic cholesterol siRNA conjugates. The cholesterol tagging technology is design on the rationale of hydrophobic siRNAs approach, but hydrophobic modulation throughout the whole siRNA backbone for efficient membrane fusion and transmembrane delivery. The challenge and potential of this technology for preclinical development are also discussed. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Neutrophil-Mediated Delivery of Dexamethasone Palmitate-Loaded Liposomes Decorated with a Sialic Acid Conjugate for Rheumatoid Arthritis Treatment.

PURPOSE: The aim of this research was to design dexamethasone palmitate (DP) loaded sialic acid modified liposomes, with the eventual goal of using peripheral blood neutrophils (PBNs) that carried drug-loaded liposomes to improve the therapeutic capacity for rheumatoid arthritis (RA). METHODS: A sialic acid - cholesterol conjugate (SA-CH) was synthesized and anchored on the surface of liposomal dexamethasone palmitate (DP-SAL). The physicochemical characteristics and in vitro cytotoxicity of liposomes were evaluated. Flow cytometry and confocal laser scanning microscopy were utilized to investigate the accumulation of liposomes in PBNs. The adjuvant-induced arthritis was adopted to investigate the targeting ability and anti-inflammatory effect of DP loaded liposomes. RESULTS: Both DP-CL and DP-SAL existed an average size less than 200 nm with remarkably high encapsulation efficiencies more than 90%. In vitro and in vivo experiments manifested SA-modified liposomes provided a reinforced accumulation of DP in PBNs. As well, DP-SAL displayed a greater degree of accumulation in the joints and a stronger anti-inflammatory effect in terms of RA suppression. CONCLUSIONS: SA-modified liposomal DP was a promising candidate for RA-targeting treatment through the neutrophil-mediated drug delivery system.

Recent preclinical and clinical advances in oligonucleotide conjugates.

INTRODUCTION: Oligonucleotide therapeutics have the potential to change the way disease is treated due to their ability to modulate gene expression of any therapeutic target in a highly specific and potent manner. Unfortunately, this drug class is plagued with inherently poor pharmacological characteristics, which need to be overcome. The development of a chemical modification library for oligonucleotides has addressed many of the initial challenges, but delivery of these payloads across plasma membranes remains difficult. The latest technological advances in oligonucleotide therapeutics utilizes direct conjugation to targeting ligands, which has improved bioavailability and target tissue exposure many-fold. The success of this approach has resulted in numerous clinical programs over the past 5 years. AREAS COVERED: We review the literature on oligonucleotide conjugate strategies which have proven effective preclinically and clinically. We summarize the chemical modifications which allow parenteral administration as well as evaluate the efficacy of a multitude of conjugate approaches including lipids, peptides, carbohydrates, and antibodies. EXPERT OPINION: The success of future conjugate strategies will likely rely on the effective combination of characteristics from earlier technologies. High-affinity ligand-receptor interactions can be critical to achieving meaningful accumulation in target tissues, but pharmacokinetic modulators which increase the circulating half-life may also be necessary. Synthesis of these approaches has the potential to bring the next breakthrough in oligonucleotide therapeutics.