Intravenous siRNA of Brain Cancer with Receptor Targeting and Avidin–Biotin Technology

Purpose The effective delivery of short interfering RNA (siRNA) to brain following intravenous administration requires the development of a delivery system for transport of the siRNA across the brain capillary endothelial wall, which forms the blood–brain barrier in vivo. Methods siRNA was delivered to brain in vivo with the combined use of a receptor-specific monoclonal antibody delivery system, and avidin–biotin technology. The siRNA was mono-biotinylated on either terminus of the sense strand, in parallel with the production of a conjugate of the targeting MAb and streptavidin. Results Rat glial cells (C6 or RG-2) were permanently transfected with the luciferase gene, and implanted in the brain of adult rats. Following the formation of intra-cranial tumors, the rats were treated with a single intravenous injection of 270 μg/kg of biotinylated siRNA attached to a transferrin receptor antibody via a biotin–streptavidin linker. The intravenous administration of the siRNA caused a 69–81% decrease in luciferase gene expression in the intracranial brain cancer in vivo. Conclusions Brain delivery of siRNA following intravenous administration is possible with siRNAs that are targeted to brain with the combined use of receptor specific antibody delivery systems and avidin–biotin technology.     

Delivery of small interfering RNAs by nanovesicles for cancer therapy

Small interfering ribonucleic acids (siRNAs) are originally recognized as an intermediate of the RNA interference (RNAi) pathway. They can inhibit or silence various cellular pathways by knocking down specific messenger RNA molecules. In cancer cells, siRNAs can suppress the expression of several multidrug-resistant genes, leading to the increased deposition of chemotherapeutic drugs at the tumor site. siRNA therapy can be used to selectively increase apoptosis of cancer cells or activate an immune response to the cancer. However, delivering siRNAs to the targeted location is the main limitation in achieving safe and effective delivery of siRNAs. This review highlights some representative examples of nonviral delivery systems, especially nanovesicles such as exosomes, liposomes, and niosomes. Nanovesicles can improve the delivery of siRNAs by increasing their intracellular delivery, and they have demonstrated excellent potential for cancer therapy. This review focuses on recent discoveries of siRNA targets for cancer therapy and the use of siRNAs to successfully silence these targets. In addition, this review summarizes the recent progress in designing nanovesicles (liposomes or niosomes) for siRNA delivery to cancer cells and the effects of a combination of anticancer drugs and siRNA therapy in cancer therapy.     

Drug Targeting to the Brain

The goal of brain drug targeting technology is the delivery of therapeutics across the blood–brain barrier (BBB), including the human BBB. This is accomplished by re-engineering pharmaceuticals to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Certain endogenous peptides, such as insulin or transferrin, undergo receptor-mediated transport (RMT) across the BBB in vivo. In addition, peptidomimetic monoclonal antibodies (MAb) may also cross the BBB via RMT on the endogenous transporters. The MAb may be used as a molecular Trojan horse to ferry across the BBB large molecule pharmaceuticals, including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene medicines. Fusion proteins of the molecular Trojan horse and either neurotrophins or single chain Fv antibodies have been genetically engineered. The fusion proteins retain bi-functional properties, and both bind the BBB receptor, to trigger transport into brain, and bind the cognate receptor inside brain to induce the pharmacologic effect. Trojan horse liposome technology enables the brain targeting of non-viral plasmid DNA. Molecular Trojan horses may be formulated with fusion protein technology, avidin–biotin technology, or Trojan horse liposomes to target to brain virtually any large molecule pharmaceutical.     

siRNA抑制人乳腺癌SK-BR-3细胞株VEGF基因表达的实验研究

目的研究siRNA(small interfering,RNA)对乳腺癌细胞SK-BR-3的VEGF基因表达的抑制作用,为RNAi技术在肿瘤生物治疗中的应用提供实验基础。方法体外合成一条针对VEGF基因的siRNA,使用脂质体转染的方法导入细胞,观察转染后乳腺癌细胞SK-BR-3的增殖变化,MTT法检测细胞存活率,RT-PCR检测转染后VEGFmRNA表达水平的变化,ELISA检测蛋白表达的下降效果。结果所设计的siRNA能有效抑制乳腺癌细胞的生长;降低了VEGFmRNA的表达;蛋白表达水平也显著降低。作为阴性对照的错义序列组siRNA则没有这种效果,不起作用。结论 siRNA可以有效抑制细胞株SK-BR-3中VEGF的表达,从而抑制细胞生长。应用RNA干扰技术可以有效抑制肿瘤细胞的增殖。     

Nano carriers for drug transport across the blood–brain barrier

Effective therapy lies in achieving a therapeutic amount of drug to the proper site in the body and then maintaining the desired drug concentration for a sufficient time interval to be clinically effective for treatment. The blood–brain barrier (BBB) hinders most drugs from entering the central nervous system (CNS) from the blood stream, leading to the difficulty of delivering drugs to the brain via the circulatory system for the treatment, diagnosis and prevention of brain diseases. Several brain drug delivery approaches have been developed, such as intracerebral and intracerebroventricular administration, intranasal delivery and blood-to-brain delivery, as a result of transient BBB disruption induced by biological, chemical or physical stimuli such as zonula occludens toxin, mannitol, magnetic heating and ultrasound, but these approaches showed disadvantages of being dangerous, high cost and unsuitability for most brain diseases and drugs. The strategy of vector-mediated blood-to-brain delivery, which involves improving BBB permeability of the drug–carrier conjugate, can minimize side effects, such as being submicrometre objects that behave as a whole unit in terms of their transport and properties, nanomaterials, are promising carrier vehicles for direct drug transport across the intact BBB as a result of their potential to enter the brain capillary endothelial cells by means of normal endocytosis and transcytosis due to their small size, as well as their possibility of being functionalized with multiple copies of the drug molecule of interest. This review provids a concise discussion of nano carriers for drug transport across the intact BBB, various forms of nanomaterials including inorganic/solid lipid/polymeric nanoparticles, nanoemulsions, quantum dots, nanogels, liposomes, micelles, dendrimers, polymersomes and exosomes are critically evaluated, their mechanisms for drug transport across the BBB are reviewed, and the future directions of this area are fully discussed.     

VEGF-mediated tight junctions pathological fenestration enhances doxorubicin-loaded glycolipid-like nanoparticles traversing BBB for glioblastoma-targeting therapy

The existence of blood–brain barrier (BBB) greatly hindered the penetration and accumulation of chemotherapeutics into glioblastoma (GBM), accompany with poor therapeutic effects. The growth of GBM supervene the impairment of tight junctions (TJs); however, the pathogenesis of BBB breakdown in GBM is essentially poorly understood. This study found that vascular endothelial growth factor (VEGF) secreted by GBM cells plays an important role in increasing the permeability of BBB by disrupting endothelial tight junction proteins claudin-5 and thus gave doxorubicin (DOX)-loaded glycolipid-like nanoparticles (Ap-CSSA/DOX), an effective entrance to brain tumor region for GBM-targeting therapy. In addition, VEGF downregulates the expression of claudin-5 with a dose-dependent mode, and interfering with the VEGF/VEGFR pathway using its inhibitor axitinib could reduce the permeability of BBB and enhance the integrity of the barrier. Ap-CSSA/DOX nanoparticles showed high affinity to expressed low-density lipoprotein receptor-related proteins 1 (LRP1) in both BBB and GBM. And BBB pathological fenestration in GBM further exposed more LRP1 binding sites for Ap-CSSA/DOX nanoparticles targeting to brain tumor, resulting in a higher transmembrane transport ratio in vitro and a stronger brain tumor biodistribution in vivo, and finally realizing a considerable antitumor effect. Overall, taking advantage of BBB pathological features to design an appropriate nanodrug delivery system (NDDS) might provide new insights into other central nervous system (CNS) diseases treatment.     

Long-Circulating PEGylated Polycyanoacrylate Nanoparticles as New Drug Carrier for Brain Delivery

Purpose. The aim of this study was to evaluate the ability of long-circulating PEGylated cyanoacrylate nanoparticles to diffuse into the brain tissue. Methods. Biodistribution profiles and brain concentrations of [¹⁴C]-radiolabeled PEG-PHDCA, polysorbate 80 or poloxamine 908-coated PHDCA nanoparticles, and uncoated PHDCA nanoparticles were determined by radioactivity counting after intravenous administration in mice and rats. In addition, the integrity of the blood-brain barrier (BBB) after nanoparticles administration was evaluated by in vivo quantification of the diffusion of [¹⁴C]-sucrose into the brain. The location of fluorescent nanoparticles in the brain was also investigated by epi-fluorescent microscopy. Results. Based on their long-circulating characteristics, PEGylated PHDCA nanoparticles penetrated into the brain to a larger extent than all the other tested formulations. Particles were localized in the ependymal cells of the choroid plexuses, in the epithelial cells of pia mater and ventricles, and to a lower extent in the capillary endothelial cells of BBB. These phenomena occurred without any modification of BBB permeability whereas polysorbate 80-coated nanoparticles owed, in part, their efficacy to BBB permeabilization induced by the surfactant. Poloxamine 908-coated nanoparticles failed to increase brain concentration probably because of their inability to interact with cells. Conclusions. This study proposes PEGylated poly (cyanoacrylate) nanoparticles as a new brain delivery system and highlights two requirements to design adequate delivery systems for such a purpose: a) long-circulating properties of the carrier, and b) appropriate surface characteristics to allow interactions with BBB endothelial cells.     

Targeted delivery systems of small interfering RNA by systemic administration

RNA interference (RNAi) is induced by 21-25 nucleotide, double-stranded small interfering RNA (siRNA), which is incorporated into the RNAi-induced silencing complex (RISC) and is a guide for cleavage of the complementary target mRNA in the cytoplasm. There are many obstacles to in vivo delivery of siRNAs, such as degradation by enzymes in blood, interaction with blood components and non-specific uptake by the cells, which govern biodistribution in the body. In order to achieve the knockdown by siRNAs in vivo, many delivery systems of siRNAs based on physical and pharmaceutical approaches have been proposed. In addition, the immune responses of siRNA must be taken into account when considering the application of siRNAs to in vivo therapy. This review focuses on recent reports about delivery systems and immune responses of siRNAs.     

Solubilization of flurbiprofen into aptamer-modified PEG–PLA micelles for targeted delivery to brain-derived endothelial cells in vitro

Abstract

Novel aptamer-functionalized polyethylene glycol–polylactic acid (PEG–PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer’s disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.     

Palmitic acid-conjugated 21-nucleotide siRNA enhances gene-silencing activity

Short interfering RNA (siRNA) technology is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with palmitic acid at the 5'-end of the sense strand (C16-siRNAs) using our novel synthesis strategy in order to improve the potency of siRNA. The C16-siRNAs exhibited enhanced nuclease stability. In addition, they showed potent gene-silencing efficacy against exogenous Renilla luciferase in HeLa cells compared with a nonmodified siRNA in the presence of Lipofectamine 2000. The C16-siRNAs also had a more potent inhibitory effect on Renilla luciferase activity than the other siRNA conjugated with lipids at the 5'-end and the 3'-end by palmitoyl conjugation. For further improvement, the gene silencing potency of the C16-siRNAs against the endogenous vascular endothelial growth factor (VEGF) gene in HeLa cells was investigated. In this investigation, the siRNAs were prepared not only with the normal RNA sequence but also coupled with an inverted thymidine (idT) at the 3'-ends of both the sense and antisense strands (siRNA-idT), including palmitic acid conjugations at the 5'-end of the sense strand, to improve stability. The C16-siRNA including idT modifications exhibited a significantly greater inhibitory effect on the VEGF gene in the presence of Lipofectamine 2000. It is noteworthy that C16-siRNA-idT demonstrated long-term gene-silencing efficacy of up to 5 days. Interestingly, the C16-siRNAs, including that with idT modifications, exhibited strong RNAi potency in the absence of any transfection reagents, although only at high concentrations. Both the C16-siRNAs and C16-siRNA-idT induced a high level of membrane permeability in HeLa cells. Our developed C16-siRNAs, particularly C16-siRNA-idT, are thus among the promising candidates for a new generation of modified siRNAs that can solve the many problems associated with siRNA technology.     

Insulin Fragments as a Carrier for Peptide Delivery Across the Blood–Brain Barrier

The possibility of using insulin (INS), which is transported into the brain by receptor-mediated transcytosis, as a peptide carrier for delivery across the blood-brain barrier (BBB) was investigated. After mice received an i.v. injection of horseradish peroxidase (HRP, M.W., 40,000) conjugated with INS, the HRP activity in the brain was higher than that after HRP injection. Since INS-HRP lowered the blood glucose level, we prepared insulin fragments by chemical and enzymatic procedures in an effort to find a carrier with no hypoglycemic activity. Seven fragments were synthesized taking the binding regions into consideration, but none showed any receptor binding affinity in cultures of bovine brain micro vessel endothelial cells (BMEC). However, the fragment (F007) obtained by trypsin digestion showed high affinity and scarcely any hypoglycemic activity in mice even at a dose ten times the effective dose of insulin. These results suggest that this fragment may be useful as a carrier to transport therapeutic peptides across the BBB.     

Transferrin receptor targeting segment T7 containing peptide gene delivery vectors for efficient transfection of brain tumor cells

Successful gene therapy for brain tumors are often limited by two important factors, the existence of blood brain barrier (BBB) and inefficient transfection of brain tumor cells. In this study, we designed a series of peptide-based gene delivery vectors decorated with T7 segment for binding the transferrin (Tf) receptors which were highly expressed on brain tumor cells, and evaluated their ability of gene delivery. The physicochemical properties of peptide vectors or peptide/DNA complexes were studied as well. The in vitro transfection efficiency was investigated in normal and glioma cell lines. Among these complexes, PT-02/DNA complexes showed the highest transfection efficiency in glioma cells and low cytotoxicity in normal cell lines, and it could transport DNA across the BBB model in vitro. Furthermore, PT-02/DNA could deliver pIRES2-EGFP into the brain site of zebrafish in vivo. The designed peptide vectors offered a promising way for glioma gene therapy.     

RNA interference as a gene-specific approach for molecular medicine

The discovery of RNA interference (RNAi) in eukaryotic cells has been the major recent breakthrough in molecular and cell biology. RNAi machineries exert biological functions in gene regulation, genome defense and chromatin architecture and dynamics. The potential of RNAi to silence any gene of interest in a highly specific and efficient manner via double-stranded RNA (dsRNA) has literally revolutionized modern genetics. RNAi-based functional genomics now permits, for the first time, to evaluate the cellular role of individual gene products on a genome-wide scale in higher organisms like mammals, presenting an alternative to the generation of animal knockouts often doomed to failure because of a lethal phenotype. RNAi has had an enormous impact on the development of novel disease models in animals, and it is likely that small interfering RNAs (siRNAs), which are the trigger molecules for RNA silencing, will become an invaluable tool for the treatment of genetic diseases. First clinical trials, using siRNAs directed against the vascular endothelial growth factor (VEGF) or one of its receptors, have been initiated recently for the treatment of age-related macular degeneration. Improving guidelines for the rational design of siRNAs, based on recent progress in understanding the mechanisms underlying RNAi, as well as the introduction of chemical modifications into siRNAs are expected to improve their pharmacokinetic and pharmacodynamic properties for in vivo applications. Finally, successful therapeutic application of RNAi will depend on the development of improved siRNA delivery strategies that combine high specificity and efficiency with a low immunostimulatory and tumorigenic potential.     

Influence of Short-Chain Cell-Penetrating Peptides on Transport of Doxorubicin Encapsulating Receptor-Targeted Liposomes Across Brain Endothelial Barrier

Purpose

To investigate the influence of different cell penetrating peptides (CPPs-TAT, Penetratin and Mastoparan), on the transport of doxorubicin encapsulating transferrin (Tf)-liposomes across brain endothelial barrier, in vitro and in vivo.

Methods

The cellular uptake of dual-functionalized, (Tf-CPP), liposomes into various tumor cells was assessed using HPLC. The transport of liposomes was also measured across a robust 3D brain tumor model constructed using chitosan-PLGA scaffolds. The growth of tumor cells was monitored using H&E staining and the fully grown tumor scaffolds were visualized using SEM. The tumor scaffolds were combined with the culture inserts carrying tightly packed brain endothelial cells. The in vitro and in vivo transport of drug (using Tf-CPP-liposomes) across the brain endothelial barrier was determined by extraction of the drug from cells and tissues followed by analysis using HPLC.

Results

The results demonstrated improved delivery of doxorubicin using dual-functionalized liposomes versus the single ligand or unmodified liposomes. Among different Tf-CPP-liposomes, the Tf-Penetratin liposomes showed efficient cellular transport of the encapsulated drug (approximately 90–98%) and maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB). The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration.

Conclusions

The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.     

适配子介导siRNA靶向递送系统的研究进展

RNA干扰技术(RNAi)作为一种新型的基因治疗技术已应用到许多疾病的体外研究中,但靶向递送技术仍是目前制约小干扰RNA(siRNA)药物成功进入临床的关键技术问题。寡核苷酸适配子具有高亲和性、高靶向性,成为递送siRNA的重要手段。本文综述了siRNA体内递送的主要障碍,以及近年来适配子介导的siRNA靶向递送系统的最新研究进展。     

Investigation of the mechanism of enhancement of central nervous system delivery of 2'-beta-fluoro-2',3'-dideoxyinosine via a blood-brain barrier adenosine deaminase-activated prodrug.

Enhanced central nervous system (CNS) delivery of certain poorly penetrating 2',3'-dideoxynucleosides has been achieved by designing prodrugs that are substrates for enzymes, such as adenosine deaminase (ADA), that are present at high activities in brain tissue. In this study, the potential role of adenosine deaminase localized within the endothelial cells of the blood-brain barrier (BBB) in providing enhanced intracellular and CNS delivery of an ADA-activated prodrug is assessed in vitro using cell culture models of the BBB. The kinetics of uptake and bioconversion of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a model ADA-activated prodrug of 2'-beta-fluoro-2',3'-dideoxyinosine, were determined in primary cultured bovine brain microvascular endothelial cells. Model-based simulations of CNS availability derived from in vitro estimates of parameters for simple passive diffusion and ADA-catalyzed deamination suggest that ADA that is localized within the BBB plays an important role in the conversion of F-ddA to 2'-beta-fluoro-2',3'-dideoxyinosine during its passage across the BBB. Consistent with in vivo observations, these simulations demonstrate that elevated levels of certain enzymes, such as ADA, in the brain microvascular endothelial cells of the BBB may be exploited in the design of brain-targeted prodrugs or drug-carrier conjugates, which brain tissue selectively converts.     

Specific Binding,Uptake, and Transport of ICAM-1-Targeted Nanocarriers Across Endothelial and Subendothelial Cell Components of the Blood–Brain Barrier

Purpose

The blood–brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models.

Methods

Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures.

Results

ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB.

Conclusions

CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.     

Brain drug delivery technologies: novel approaches for transporting therapeutics

The blood-brain barrier (BBB) denies many therapeutic agents access to brain tumours and other diseases of the central nervous system (CNS). Despite remarkable advances in our understanding of the mechanisms involved in the development of the brain diseases and the actions of neuroactive agents, drug delivery to the brain remains a challenge. For more than 20 years, extensive efforts have been made to enhance delivery of therapeutic molecules across vascular barriers of the CNS. The current challenge is to develop drug-delivery strategies that will allow the passage of drug molecules through the BBB in a safe and effective manner, and this review will provide an insight into some of the strategies developed to enhance drug delivery across the BBB.     

Brain Endothelial Cell-Cell Junctions: How to “Open” the Blood Brain Barrier

The blood-brain barrier (BBB) is a highly specialized structural and biochemical barrier that regulates the entry of blood-borne molecules into brain, and preserves ionic homeostasis within the brain microenvironment. BBB properties are primarily determined by junctional complexes between the cerebral endothelial cells. These complexes are comprised of tight and adherens junctions. Such restrictive angioarchitecture at the BBB reduces paracellular diffusion, while minimal vesicle transport activity in brain endothelial cells limits transcellular transport. Under normal conditions, this largely prevents the extravasation of large and small solutes (unless specific transporters are present) and prevents migration of any type of blood-borne cell. However, this is changed in many pathological conditions. There, BBB disruption (“opening”) can lead to increased paracellular permeability, allowing entry of leukocytes into brain tissue, but also contributing to edema formation. In parallel, there are changes in the endothelial pinocytotic vesicular system resulting in the uptake and transfer of fluid and macromolecules into brain parenchyma. This review highlights the route and possible factors involved in BBB disruption in a variety of neuropathological disorders (e.g. CNS inflammation, Alzheimer’s disease, Parkinson’s disease, epilepsy). It also summarizes proposed signal transduction pathways that may be involved in BBB “opening”.