anti-microRNA Oligonucleotides Research Articles

Pulmonary delivery of anti-microRNA oligonucleotide and glycyrrhizic acid using ternary peptide micelles for the treatment of acute lung injury

Pulmonary delivery of anti-microRNA oligonucleotide and glycyrrhizic acid using ternary peptide micelles for the treatment of acute lung injury

Inhibition of microRNA-33b specifically ameliorates abdominal aortic aneurysm formation via suppression of inflammatory pathways

Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA.

Structure-Activity Relationships of Anti-microRNA Oligonucleotides Containing Cationic Guanidine-Modified Nucleic Acids.

Anti-microRNA oligonucleotides (AMOs) are valuable tools for the treatment of diseases caused by the dysregulation of microRNA expression. However, the correlation between chemical modifications in AMO sequences and the microRNA-inhibitory activity has not been fully elucidated. In this study, we synthesized a series of AMOs containing cationic guanidine-bridged nucleic acids (GuNA) and evaluated their activities using a dual luciferase assay. We also optimized the site of GuNA substitution and found an effective design for the inhibition of microRNA-21, which was partially different from that of conventional nucleic acid derivatives. This study showed that GuNA-substituted AMOs are effective in inhibiting the function of microRNA.

Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides.

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

Hypoxia-specific anti-RAGE exosomes for nose-to-brain delivery of anti-miR-181a oligonucleotide in an ischemic stroke model.

Ischemic stroke is caused by a reduction in blood flow to the brain due to narrowed cerebral arteries. Thrombolytic agents have been used to induce reperfusion of occluded cerebral arteries. However, brain damage continues to progress after reperfusion and induces ischemia-reperfusion (I/R) injury. The receptor for advanced glycation end-products (RAGE) is overexpressed in hypoxic cells of the ischemic brain. In this study, an exosome linked to RAGE-binding-peptide (RBP-Exo) was developed as a hypoxia-specific carrier for nose-to-brain delivery of anti-microRNA oligonucleotide (AMO). The RBP-Exos were less than 50 nm in size and had negative surface charge. In vitro studies showed that RBP-Exos delivered AMO181a to Neuro2A cells more efficiently than unmodified exosomes (Unmod-Exos). In addition, RAGE was downregulated by RBP-Exos, suggesting that the RBP moiety of the RBP-Exos reduced the RAGE-mediated signal pathway. MicroRNA-181a (miR-181a) is one of the upregulated miRNAs in the ischemic brain and its downregulation can reduce the damage to the ischemic brain. Cholesterol-modified AMO181a (AMO181a-chol) was loaded onto the RBP-Exo by hydrophobic interaction. The AMO181a-chol-loaded RBP-Exo (RBP-Exo/AMO181a-chol) was administered intranasally to a rat middle cerebral artery occlusion (MCAO) model. MiR-181a was knocked down and Bcl-2 was upregulated by intranasal delivery of RBP-Exo/AMO181a-chol. In addition, tumor necrosis factor-α (TNF-α) expression and apoptosis were reduced by RBP-Exo/AMO181a-chol. As a result, RBP-Exo/AMO181a-chol significantly suppressed infarct size compared with the controls. In conclusion, RBP-Exo was a hypoxia-specific carrier for nose-to-brain delivery of AMO181a-chol in an ischemic stroke model. Furthermore, the combined effects of RBP and AMO181a-chol exerted neuroprotective effects in the ischemic brain.

Ester modification at the 3′ end of anti-microRNA oligonucleotides increases potency of microRNA inhibition

MicroRNAs (miRNAs) are short noncoding RNAs that play a fundamental role in gene regulation. Deregulation of miRNA expression has a strong correlation with disease and antisense oligonucleotides that bind and inhibit miRNAs associated with disease have therapeutic potential. Current research on the chemical modification of anti-miRNA oligonucleotides (anti-miRs) is focused on alterations of the phosphodiester-ribose backbone to improve nuclease resistance and binding affinity to miRNA strands. Here we describe a structure-guided approach for modification of the 3′-end of anti-miRs by screening for modifications compatible with a nucleotide-binding pocket present on human Argonaute2 (hAgo2). We computationally screened a library of 190 triazole-modified nucleoside analogs for complementarity to the t1A-binding pocket of hAgo2. Seventeen top scoring triazoles were then incorporated into the 3′ end of anti-miR21 and potency was evaluated for each in a cell-based assay for anti-miR activity. Four triazole-modified anti-miRs showed higher potency than anti-miR21 bearing a 3′ adenosine. In particular, a triazole-modified nucleoside bearing an ester substituent imparted a nine-fold and five-fold increase in activity for both anti-miR21 and anti-miR122 at 300 and 5 nM, respectively. The ester group was shown to be critical as a similar carboxylic acid and amide were inactive. Furthermore, anti-miR 3′ end modification with triazole-modified nucleoside analogs improved resistance to snake venom phosphodiesterase, a 3′-exonuclease. Thus, the modifications described here are good candidates for improvement of anti-miR activity.

Approaches to Modulate the Chronic Wound Environment Using Localized Nucleic Acid Delivery.

Significance: Nonhealing wounds have been the subject of decades of basic and clinical research. Despite new knowledge about the biology of impaired wound healing, little progress has been made in treating chronic wounds, leaving patients with few therapeutic options. Diabetic ulcers are a particularly common form of nonhealing wound. Recent Advances: Recently, investigation of therapeutic nucleic acids (TNAs), including plasmid DNA, small interfering RNA, microRNA mimics, anti-microRNA oligonucleotides, messenger RNA, and antisense oligonucleotides, has created a new treatment strategy for chronic wounds. TNAs can modulate the wound toward a prohealing environment by targeting gene pathways associated with inflammation, proteases, cell motility, angiogenesis, epithelialization, and oxidative stress. A variety of delivery systems have been investigated for TNAs, including dendrimers, lipid nanoparticles (NPs), polymeric micelles, polyplexes, metal NPs, and hydrogels. This review summarizes recent developments in TNA delivery for therapeutic targets associated with chronic wounds, with an emphasis on diabetic ulcers. Critical Issues: Translational potential of TNAs remains a key challenge; we highlight some drug delivery approaches for TNAs that may hold promise. We also describe current commercial efforts to locally deliver nucleic acids to modulate the wound environment. Future Directions: Localized nucleic acid delivery holds promise for the treatment of nonhealing chronic wounds. Future efforts to improve targeting of these nucleic acid therapies in the wound with both spatial and temporal control through drug delivery systems will be crucial to successful clinical translation.

Pharmacokinetic Studies of Antisense Oligonucleotides Using MALDI-TOF Mass Spectrometry.

Cardiac diseases are the most frequent causes of death in industrialized countries. Pathological remodeling of the heart muscle is caused by several etiologies such as prolonged hypertension or injuries that can lead to myocardial infarction and in serious cases also the death of the patient. The micro-RNA miR-132 has been identified as a master-switch in the development of cardiac hypertrophy and adverse remodeling. In this study, MALDI-TOF mass spectrometry (MS) was utilized to establish a robust and fast method to sensitively detect and accurately quantify anti-microRNA (antimiR) oligonucleotides in blood plasma. An antimiR oligonucleotide isolation protocol containing an ethanol precipitation step with glycogen as oligonucleotide carrier as well as a robust and reproducible MS-analysis procedure has been established. Proteinase K treatment was crucial for releasing antimiR oligonucleotides from plasma- as well as cellular proteins and reducing background derived from biological matrices. AntimiR oligonucleotide detection was achieved from samples of studies in different animal models such as mouse and pig where locked nucleic acids-(LNA)-modified antimiR oligonucleotides have been used to generate pharmacokinetic data.

Inhibition of breast cancer cell proliferation with anti-microRNA oligonucleotides flanked by interstrand cross-linked duplexes

Breast cancer is the most frequent cancer affecting women worldwide. Traditional chemotherapy, hormone therapy, and targeted therapy are used for breast cancer treatment. However, breast cancer is a heterogeneous disease, and patients often develop drug resistance. Therefore, various new therapeutic strategies have been investigated, including microRNA regulation. Anti-microRNA oligonucleotides (AMOs) are one of the most potent agents in oligonucleotide therapy. The inhibition activity of an AMO can be increased by flanking its single-stranded antisense sequence (the widely used structure for AMOs) with interstrand cross-linked duplexes (CLDs). An extrastable CLD improves nuclease resistance and stabilizes hybridization with a target. This study investigated the effects of anti-microRNA-21 (miR-21) AMO modified with CLDs on breast cancer cells without using reporter assay. The CLD-modified AMO suppressed breast cancer cell proliferation for a long duration compared to other types of AMOs. In addition, it expectedly up-regulated the miR-21-controlled expression of tumor suppressor genes. Therefore, an AMO flanked by CLDs can be a promising strategy for breast cancer treatment.

Co-administration of Anti microRNA-124 and -137 Oligonucleotides Prevents Hippocampal Neural Stem Cell Loss Upon Non-convulsive Seizures.

Convulsive seizures promote adult hippocampal neurogenesis (AHN) through a transient activation of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). However, in a significant population of epilepsy patients, non-convulsive seizures (ncSZ) are observed. The response of NSPCs to non-convulsive seizure induction has not been characterized before. We here studied first the short-term effects of controlled seizure induction on NSPCs fate and identity. We induced seizures of controlled intensity by intrahippocampally injecting increasing doses of the chemoconvulsant kainic acid (KA) and analyzed their effect on subdural EEG recordings, hippocampal structure, NSPC proliferation and the number and location of immature neurons shortly after seizure onset. After establishing a KA dose that elicits ncSZ, we then analyzed the effects of ncSZ on NSPC proliferation and NSC identity in the hippocampus. ncSZ specifically triggered neuroblast proliferation, but did not induce proliferation of NSPCs in the SGZ, 3 days post seizure onset. However, ncSZ induced significant changes in NSPC composition in the hippocampus, including the generation of reactive NSCs. Interestingly, intrahippocampal injection of a combination of two anti microRNA oligonucleotides targeting microRNA-124 and -137 normalized neuroblast proliferation and prevented NSC loss in the DG upon ncSZ. Our results show for the first time that ncSZ induce significant changes in neuroblast proliferation and NSC composition. Simultaneous antagonism of both microRNA-124 and -137 rescued seizure-induced alterations in NSPC, supporting their coordinated action in the regulation of NSC fate and proliferation and their potential for future seizure therapies.

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes.

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3′ UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2′-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5′ and 3′ termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3′-side CL improves nuclease resistance, whereas the 5′-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3′ part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Different roles of a novel shrimp microRNA in white spot syndrome virus (WSSV) and Vibrio alginolyticus infection

In this study, Marsupeneaus japonicus microRNA-S5 (miR-S5) was found to be up-regulated 24 h post white spot syndrome virus (WSSV) or V. alginolyticus infection. The loss of function using an anti-microRNA oligonucleotide (AMO-miR-S5) showed that expression levels of multiple innate immune-related genes were affected. The expression of p53 and tumor necrosis factor-α (TNF-α) were significantly down-regulated, expression of myosin was significantly up-regulated. The miR-S5 knockdown delayed WSSV-induced death for 48 h, but the final mortality was not affected, while V. alginolyticus-induced mortality was increased by 30%. The effect of miR-S5 knockdown on phagocytosis and apoptosis rates showed that miR-S5 knock down significantly decreased phagocytosis rate of WSSV from 27.8% to 7.0%, and phagocytosis rate of V. alginolyticus from 27.2% to 21.4%, separately. WSSV-induced apoptosis decreased from 60.83% to 51.25%, but no effect on V. alginolyticus-induced apoptosis (43.72%–45.04%). We concluded that miR-S5 could be used by WSSV via regulating hemocyte phagocytosis and apoptosis processes, but helps to defend against bacterial infection by regulating the proPO system, superoxide dismutase activity and phagocytosis.

Quantitative relationship between chemical properties and bioactivities of anti-microRNA oligonucleotides targeted to tumor-associated microRNA-21

Synthetic anti-microRNA oligonucleotides (AMOs) are promising drug candidates to inactivate disease-related microRNAs because of their sequence-specific binding to their targets and the variety of chemical modifications available. Over the last decade, the qualitative relationships between the chemical properties of AMOs and bioactivity (inactivation of their target miRNAs) have been studied to enhance their bioactivity. On the other hand, in real-world drug development, drugs must be designed case-by-case, taking many factors into account. Thus, in order to design AMOs that target specific miRNA, understanding the quantitative relationship between the chemical properties of AMOs and inactivation of their target miRNA is necessary. Here, we aimed to find the specific quantitative relationship of AMOs targeted to tumor-associated miR-21 through direct comparison of their inactivation efficacies with systematically varied chemical properties, including sequence-specific binding affinity, nuclease resistance, and RNase H activation. As a result, we newly found the quantitative relationships; (1) sequence-specific binding affinity of AMOs against miR-21 is the main determining factor for inactivation efficacy, (2) nuclease resistance of AMOs impacts their miR-21 inactivation efficacy acting cooperatively with the binding affinity, although nuclease resistance alone does not affect the miRNA inactivation efficacy, and (3) RNase H activation is unnecessary. This study also demonstrates the utility of the obtained relationship for the design of AMO-based drugs targeted to miR-21, through cell-based analyses. Thus, the obtained quantitative relationship would make it possible to predict the miR-21 inactivation efficacy of AMOs which are newly designed.

Renal Delivery of Anti-microRNA Oligonucleotides in Rats.

MicroRNAs are endogenous small, non-protein-coding RNA molecules that play an important role in the regulation of a wide variety of cellular functions and disease processes. A novel role for microRNAs in the development of hypertension and hypertensive tissue injury is emerging in recent studies. Development of hypertension involves multiple organ systems and cannot be modeled in vitro. Therefore, the ability to experimentally alter genes, gene products, or biological pathways, including microRNAs, in an organ-specific manner in intact animal models is particularly valuable to hypertension research. The kidney plays a central role in the long-term regulation of arterial blood pressure. In this chapter, we describe a detailed protocol for using a renal interstitial injection method to deliver anti-miR oligonucleotides to knock down microRNA specifically in the kidney in conscious rats.

Non-nucleotide Modification of Anti-miRNA Oligonucleotides.

MicroRNAs (miRNAs) are important modulators of gene expression. Synthetic anti-microRNA oligonucleotides (AMOs, or anti-miRs) are a form of steric-blocking antisense oligonucleotides (ASOs) that inhibit miRNA function through high-affinity binding and subsequent inactivation and/or degradation of the targeted miRNA. AMOs are a primary tool used to empirically determine the biological targets of a miRNA and can also be used therapeutically when overexpression of a miRNA contributes to a disease state. Chemical modification of synthetic AMOs enhance potency by protecting the oligonucleotide from nuclease degradation and by increasing binding affinity to the target miRNA. A new steric-blocking ASO modification strategy with favorable properties for use in AMOs was recently developed that combines use of high-affinity 2'-O-methyl RNA with terminally positioned non-nucleotide "ZEN" modifiers. This protocol describes use of ZEN AMOs in a dual-luciferase reporter assay as a simplified means to validate AMO performance or to quickly test putative miRNA binding sites in target sequences. This protocol also describes a method using Western blot analysis for quantifying the level of upregulation of proteins made from an mRNA that is thought to be under miRNA regulation, following inhibition of that miRNA by ZEN AMO treatment.

The miR-17∼92 cluster contributes to MLL leukemia through the repression of MEIS1 competitor PKNOX1

Mixed lineage leukemias have a relatively poor prognosis and arise as a result of translocations between the MLL(KMT2A) gene and one of multiple partner genes. Downstream targets of MLL are aberrantly upregulated and include the developmentally important HOX genes and MEIS1, as well as multiple microRNAs (miRNAs), including the miR-17∼92 cluster. Here we examined the contribution of specific miRNAs to MLL leukemias through knockdown studies utilizing custom anti-microRNA oligonucleotides. Combinatorial treatment against miR-17-5p and miR-19a-3p of the miR-17∼92 cluster dramatically reduces colony forming ability of MLL-fusion containing cell lines relative to non-MLL acute myeloid leukemia (AML) controls. To determine the mechanism by which these miRNAs contribute to leukemia, we validated PKNOX1 as a target of both miR-17-5p and miR-19a-3p. MEIS1 and PKNOX1 are TALE domain proteins that participate in ternary complexes with HOX and PBX partners. Here we establish the competitive relationship between PKNOX1 and MEIS1 in PBX-containing complex formation and determine the antagonistic role of PKNOX1 to leukemia in a murine MLL-AF9 model. These data implicate the miR-17∼92 cluster as part of a regulatory mechanism necessary to maintain MEIS1/HOXA9 −mediated transformation in MLL leukemia, indicating that targeting multiple non-homologous miRNAs may be utilized as a novel therapeutic regimen.

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.

Harnessing the Power of Posttranscriptional Gene Silencing in Crohn's Disease.

Posttranscriptional gene silencing or RNA interference is a mechanism by which the expression of one or more genes is partially or fully suppressed by noncoding RNAs, particularly small RNAs. In the laboratory this concept has been utilized to introduce synthetic anti-microRNA (miRNA) oligonucleotides to downregulate or completely abrogate gene expression. MiRNAs are a group of small (~22 nucleotide), noncoding RNAs that confer such posttranscriptional regulation of gene expression.1 They have emerged as key regulators of a wide variety of biological processes and also as candidate therapeutic targets. MiRNAs silence genes by binding to target sites found within the 3′untranslated region (UTR) of the targeted mitochondrial RNA. This results in the suppression of protein synthesis and/or degradation of the transcript. A number of miRNAs have already been identified as regulators of pathways that underlie the pathogenesis of Crohn's Disease (CD).2 For example, miR-192, miR-122, miR-29, and miR-146a have been shown to target and repress NOD2, which has been implicated in CD.3, 4, 5, 6 Using small RNA-sequencing a suite of miRNAs were identified in colon tissue that stratify CD patients according to disease behavior independent of the effect of inflammation. Furthermore, levels of specific miRNAs in these patients could predict progression to penetrating and fistulizing CD.7 The purpose of this short review is to highlight the advances in using posttranscriptional gene silencing in understanding and treating CD.8, 9

Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo

Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2'-O-methyl (2'-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.

A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery.

In this study, a new type of amphiphilic cetylated polyethyleneimine (PEI) was synthesized, and then polylactic-co-glycolic acid (PLGA)/cetylated PEI/hyaluronic acid nanoparticles (PCPH NPs) were developed by self-assembly as a novel type of gene-delivering vehicle. The PCPH NPs showed good DNA-condensation ability by forming polyplexes with small particle size and positive zeta potential. The transfection efficiency and cytotoxicity of PCPH NPs were evaluated as plasmid DNA vectors to transfect HepG2 in vitro. PCPH NPs exhibited much lower cytotoxicity and higher gene-transfection efficiency than PEI (25,000) and commercial transfection reagents. Furthermore, PCPH NPs were used as an anti-miR-221 vector for transfecting HepG2 cells, and anti-miR-221 was effectively transfected into cells and produced a greater inhibitory effect on cancer-cell growth by PCPH NPs. These results demonstrate that PCPH NPs can be a promising nonviral vector for gene-delivery systems.