Polymeric nanoparticles for gene delivery

Since the evolution of the concept of gene therapy, delivering therapeutic genes to the diseased cells has been a major challenge. Although viral vectors have been shown to be efficient in delivering genes, the issue of their safety is still to be solved. Meanwhile, the field of developing nonviral expression vectors has seen considerable progress. As compared with viruses, these are relatively safe but are confronted with the problem of poor transfection efficiency. With the growing understanding of the biology of gene transfection, and the continued efforts at enhancing the efficiency of nonviral expression vectors, it could soon become a preferred option for human gene therapy. In this review, the potential of polymeric nanoparticles as a gene expression vector is discussed. Furthermore, the importance of understanding the pathophysiology of disease conditions in developing gene expression vectors is discussed in Section 6.     

Non-condensing polymeric nanoparticles for targeted gene and siRNA delivery

Gene therapy has shown a tremendous potential to benefit patients in a variety of disease conditions. However, finding a safe and effective systemic delivery system is the major obstacle in this area. Although viral vectors showed promise for high transfection rate, the immunogenicity associated with these systems has hindered further development. As an alternative to viral gene delivery, this review focuses on application of novel safe and effective non-condensing polymeric systems that have shown high transgene expression when administered systemically or by the oral route. Type B gelatin-based engineered nanocarriers were evaluated for passive and active tumor-targeted delivery and transfection using both reporter and therapeutic plasmid DNA. Additionally, we have shown that nanoparticles-in-microsphere oral system (NiMOS) can efficiently deliver reporter and therapeutic gene constructs in the gastrointestinal tract. Additionally, there has been a significant recent interest in the use small interfering RNA (siRNA) as a therapeutic system for gene silencing. Both gelatin nanoparticles and NiMOS have shown activity in systemic and oral delivery of siRNA, respectively.     

Polymeric carrier systems for siRNA delivery

RNA interference is a technique to induce sequence-specific gene silencing, but is hampered by inefficient delivery of its mediator, short interfering RNA, into target cells. This review describes recent advances in siRNA delivery using polymeric carrier systems. Structural variations that have been applied to these polymers for optimizing their intracellular trafficking are discussed, as well as strategies for stabilization and targeting to diseased tissues in vivo. Recent findings have highlighted safety issues that need to be taken into account in the design of nanoparticles for clinical application.     

LNA-antisense rivals siRNA for gene silencing

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications.     

Efficient in vitro siRNA delivery and intramuscular gene silencing using PEG-modified PAMAM dendrimers

Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the results from the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and GFP transgenic mice.     

Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. However, its application was greatly hampered by the rapid degradation and poor cellular uptake. Recently, chitosan (CS) and its derivant have been considered as a promising siRNA transporter with the advantages of low toxicity, good biodegradability and biocompatibility. Chitosan of different molecular weight (Mw) and degrees of deacetylation (DD) showed significantly varied target gene silencing efficacy, and it is still not well clarified how these characteristics influence CS mediated siRNA transfection. To compare the aspects of cell permeability and intracellular unpacking of CS/siRNA complex on the effect of CS/siRNA transfection. A radiolabeled siRNA, targeting firefly luciferase gene, was loaded by chitosan of different molecular weight (varying from 2000 to 800,000 Da) and subjected to the transfection against MDA-MB-231/Luc human breast cancer cell line which stably expressed knocked in firefly Luciferase reporter gene. Following transfection intracellular radioactivity was measured to represent cell entrance ability of the CS/siRNA, while, luciferase activity in the cell lysate was also determined to reflect target gene silencing effect. The results revealed that although low molecular weight chitosan (LMWC) condensed siRNA has the highest cell permeability of almost two folds of medium molecular weight chitosan and lipofactamine, its target gene silencing effect is really low of almost eight times less than lipofectamine. This conspicuous contradiction gave us the hypothesis that LMWC generated more condensed CS/siRNA complex to facilitate cell entrance but the tight electrostatic interaction probably limited intracellular siRNA unpacking as well and unfavorably hindered target gene silencing as the final consequence. To approve this hypothesis a phosphorylatable short peptide conjugated LMWC was adopt to promote intracellular siRNA unpacking. Which was demonstrated of perfect target gene knock down ability to the extent of being even superior to lipofactamine 2000. In a conclusion, low molecular weight chitosan has the great potential to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.     

Polymeric vectors for ocular gene delivery

Gene therapy holds promise for the treatment of many inherited and acquired diseases of the eye. Successful ocular gene therapy interventions depend on efficient gene transfer to targeted cells with minimal toxicity. A major challenge is to overcome both intracellular and extracellular barriers associated with ocular gene delivery. Numerous viral and nonviral vectors were explored to improve transfection efficiency. Among nonviral delivery systems, polymeric vectors have gained significant attention in recent years owing to their nontoxic and non-immunogenic nature. Polyplexes or nanoparticles can be prepared by interaction of cationic polymers with DNA, which facilitate cellular uptake, endolysosomal escape and nuclear entry through active mechanisms. Chemical modification of these polymers allows for the generation of flexible delivery vectors with desirable properties. In this article several synthetic and natural polymeric systems utilized for ocular gene delivery are discussed.     

Recent advances of siRNA delivery by nanoparticles

Introduction: The field of RNA interference technology has been researched extensively in recent years. However, the development of clinically suitable, safe and effective drug delivery vehicles is still required.

Areas covered: This paper reviews the recent advances of non-viral delivery of small interfering RNA (siRNA) by nanoparticles, including biodegradable nanoparticles, liposomes, polyplex, lipoplex and dendrimers. The characteristics, composition, preparation, applications and advantages of different nanoparticle delivery strategies are also discussed in detail, along with the recent progress of non-viral nanoparticle carrier systems for siRNA delivery in preclinical and clinical studies.

Expert opinion: Non-viral carrier systems, especially nanoparticles, have been investigated extensively for siRNA delivery, and may be utilized in clinical applications in the future. So far, a few preliminary clinical trials of nanoparticles have produced promising results. However, further research is still required to pave the way to successful clinical applications. The most important issues that need to be focused on include encapsulation efficiency, formulation stability of siRNA, degradation in circulation, endosomal escape and delivery efficiency, targeting, toxicity and off-target effects. Pharmacology and pharmacokinetic studies also present another great challenge for nanoparticle delivery systems, owing to the unique nature of siRNA oligonucleotides compared with small molecules.     

Recent advances of siRNA delivery by nanoparticles

INTRODUCTION: The field of RNA interference technology has been researched extensively in recent years. However, the development of clinically suitable, safe and effective drug delivery vehicles is still required. AREAS COVERED: This paper reviews the recent advances of non-viral delivery of small interfering RNA (siRNA) by nanoparticles, including biodegradable nanoparticles, liposomes, polyplex, lipoplex and dendrimers. The characteristics, composition, preparation, applications and advantages of different nanoparticle delivery strategies are also discussed in detail, along with the recent progress of non-viral nanoparticle carrier systems for siRNA delivery in preclinical and clinical studies. EXPERT OPINION: Non-viral carrier systems, especially nanoparticles, have been investigated extensively for siRNA delivery, and may be utilized in clinical applications in the future. So far, a few preliminary clinical trials of nanoparticles have produced promising results. However, further research is still required to pave the way to successful clinical applications. The most important issues that need to be focused on include encapsulation efficiency, formulation stability of siRNA, degradation in circulation, endosomal escape and delivery efficiency, targeting, toxicity and off-target effects. Pharmacology and pharmacokinetic studies also present another great challenge for nanoparticle delivery systems, owing to the unique nature of siRNA oligonucleotides compared with small molecules.     

Significance and applications of nanoparticles in siRNA delivery for cancer therapy

RNAi is a powerful gene silencing process that holds great promise in cancer therapy by the use of siRNA. The aim of this review is to give an outline on different approaches to deliver siRNA and to describe the advantages and disadvantages of these systems. The prospects for siRNA are to be substantially better than other therapies, as they are easily applicable to any therapeutic target. They also promise potent gene inhibition with exquisite selectivity, down to the level of a single nucleotide polymorphism, and can easily identify offending proteins or variants by screening across a gene sequence. The main obstacle of using RNAi technology in cancer treatment is to protect such a fragile and quickly metabolized biological molecule and to efficiently deliver it in vivo to the target cells. Therefore, there is a requirement for new systems, such as nanoparticles, for siRNA delivery to help the siRNAs reach, and improve their biodistribution in, target tissues.     

Polymeric nanoparticles for drug delivery to the central nervous system

The central nervous system (CNS) poses a unique challenge for drug delivery. The blood-brain barrier significantly hinders the passage of systemically delivered therapeutics and the brain extracellular matrix limits the distribution and longevity of locally delivered agents. Polymeric nanoparticles represent a promising solution to these problems. Over the past 40years, substantial research efforts have demonstrated that polymeric nanoparticles can be engineered for effective systemic and local delivery of therapeutics to the CNS. Moreover, many of the polymers used in nanoparticle fabrication are both biodegradable and biocompatible, thereby increasing the clinical utility of this strategy. Here, we review the major advances in the development of polymeric nanoparticles for drug delivery to the CNS.     

Polymeric nano- and microparticle technologies for oral gene delivery

Gene therapy refers to local or systemic administration of a nucleic acid construct that can prevent, treat and even cure diseases by changing the expression of genes that are responsible for the pathological condition. Oral gene therapy has significant promise for treatment of local diseases such as inflammatory bowel disease and for systemic absorption of the expressed protein therapeutics. In addition, efficient oral delivery of DNA vaccines can have significant impact in disease prevention. The use of polymeric gene delivery vectors promises the translation of this experimental medical concept into clinical reality. This review addresses the challenges and opportunities in the development of polymer-based nano- and microparticle technologies for oral gene therapy. Specifically, the discussion is focused on different synthetic and natural polymers used for formulating nano- and microparticle technologies and the use of these delivery systems for oral DNA administration for therapeutic and vaccination purposes.     

Polymeric nano- and microparticle technologies for oral gene delivery

Gene therapy refers to local or systemic administration of a nucleic acid construct that can prevent, treat and even cure diseases by changing the expression of genes that are responsible for the pathological condition. Oral gene therapy has significant promise for treatment of local diseases such as inflammatory bowel disease and for systemic absorption of the expressed protein therapeutics. In addition, efficient oral delivery of DNA vaccines can have significant impact in disease prevention. The use of polymeric gene delivery vectors promises the translation of this experimental medical concept into clinical reality. This review addresses the challenges and opportunities in the development of polymer-based nano- and microparticle technologies for oral gene therapy. Specifically, the discussion is focused on different synthetic and natural polymers used for formulating nano- and microparticle technologies and the use of these delivery systems for oral DNA administration for therapeutic and vaccination purposes.     

表面修饰的LPC纳米粒介导的siRNA肿瘤靶向递送(英文)

本研究构建了能够靶向肿瘤的新型纳米粒(脂质体-鱼精蛋白-硫酸软骨素纳米粒,LPC-NP)。该纳米粒粒径约90 nm,zata电位约+35 mV。采用后插法对LPC纳米粒进行DSPE-PEG_(2000)或DSPE-PEG_(2000)-T7修饰。T7是与转铁蛋白功能类似的七肽,能够靶向转铁蛋白受体过度表达的乳腺癌细胞MCF-7。PEG修饰可显著降低血清对LPC纳米粒的聚集作用,T7修饰的纳米粒显著提高siRNA的细胞摄取和基因沉默效率。体外细胞毒实验表明抗EGFR siRNA显著抑制MCF-7细胞生长。实验结果表明经T7肽修饰的LPC纳米粒有望成为RNA干扰肿瘤治疗的递送载体。     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

siRNA for gene silencing: a route to drug target discovery

The identification of RNA interference in mammalian cells, mediated via both virally-derived short interference RNA (siRNA) and endogenously produced microRNA, has revolutionised our understanding of the translational control of gene expression. Indeed, since its initial discovery, siRNA has been rapidly deployed for the elucidation of gene function and the identification of potential drug targets, a process often known as target discovery. In this review, we briefly discuss the mechanism of RNA interference and then critically examine the use of siRNA in target discovery, with a particular emphasis upon issues such as efficacy, selectivity, delivery and application in high-throughput studies.     

Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by synthetic small interfering RNA (siRNA), and is utilized in a wide range of fields including cancer gene therapy by down-regulating a specific target protein. In this study, for tumor-targeted siRNA delivery, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated tumor gene therapy in human nasopharyngeal KB cells, which overexpressed folate receptor (FR). NP-F was composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol), Tween 80 and folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine conjugate (f-PEG(2000)-DSPE), and NP-P was substituted f-PEG(2000)-DSPE in NP-F PEG(2000)-DSPE for a non-targeting nanoparticle. The NP-F and siRNA complex (nanoplex) formed at a charge ratio (+/-) of 2/1 in the presence of 5mM NaCl was injectable size and increased transfection efficiency in the cells. NP-F showed a significantly higher intracellular amount of siRNA and stronger localization of siRNA in the cytoplasm than NP-P. When Her-2 siRNA was transfected into cells by NP-F and NP-P, NP-F significantly inhibited tumor growth, and selectively suppressed Her-2 protein expression more than NP-P. In in vivo gene therapy, a NP-F nanoplex of Her-2 siRNA by intratumoral injection significantly inhibited tumor growth of KB xenografts compared with control siRNA, but a NP-P nanoplex did not. These results of the experiments have provided optimal conditions to form folate-linked nanoparticle complexes with siRNA for folate-targeted gene therapy.