Efficient Self‐Assembled DNA Nanoparticles through Rolling Circle Amplification for siRNA Delivery in vitro

Effective and low toxicity delivery of siRNA is of great importance for clinical gene therapy. Herein, self‐assembled DNA nanoparticles (NPs) based on rolling circle amplification (RCA) with a small interfering RNA (siRNA) payload were successfully developed as a facile and efficient siRNA delivery strategy. This intracellular gene silencing strategy exhibits various advantages including low toxicity, high efficiency, and good stability. The synthesized DNA NPs serve as siRNA carriers, protecting the siRNA against nuclease degradation. We demonstrate that the obtained self‐assembled siRNA/NP/PEI system can successfully deliver enhanced green fluorescent protein (EGFP)‐siRNA into HeLa cells, realizing the same EGFP knockdown efficiency with less toxicity as that of commercial Lipofectamine 2000.     

Hybrid materials: Magnetite-Polyethylenimine-Montmorillonite, as magnetic adsorbents for Cr(VI) water treatment

Hybrid materials formed by the combination of a sodium rich Montmorillonite (MMT), with magnetite nanoparticles (40nm, Fe(3)O(4) NPs) coated with Polyethylenimine polymer (PEI 800g/mol or PEI 25000g/mol) were prepared. The intercalation of the magnetite nanoparticles coated with PEI among MMT platelets was achieved by cationic exchange. The resulting materials presented a high degree of exfoliation of the MMT sheets and a good dispersion of Fe(3)O(4) NPs on both the surface and among the layers of MMT. The presence of amine groups in the PEI structure not only aids the exfoliation of the MMT layers, but also gives to the hybrid material the necessary functionality to interact with heavy metals. These hybrid materials were used as magnetic sorbent for the removal of hexavalent chromium from water. The effect that pH, Cr(VI) concentration, and adsorbent material composition have on the Cr(VI) removal efficiency was studied. A complete characterization of the materials was performed. The hybrid materials showed a slight dependence of the removal efficiency with the pH in a wide range (1-9). A maximum amount of adsorption capacity of 8.8mg/g was determined by the Langmuir isotherm. Results show that these hybrid materials can be considered as potential magnetic adsorbent for the Cr(VI) removal from water in a wide range of pH.     

PEGylated polyethyleneimine grafted silica nanoparticles: enhanced cellular uptake and efficient siRNA delivery

The present paper reports the utilization of hybrid nanocomposite particles consisting of PEI25k-PEG5k copolymer grafted silica nanoparticles (SiO₂NPs) for enhanced cellular uptake and siRNA delivery. High-resolution transmission electron microscopy and dynamic light scattering measurements ensured the average particle size of the final hybrid component as 45 nm (core SiO₂, 28–30 nm and shell PEI25k-PEG5k, 12–15 nm). Surface morphology from atomic force microscopy analysis showed the significant relationship between the particle size and shape. ²⁹Si and ¹³C cross-polarization–magic angle spinning solid state nuclear magnetic resonance (NMR), ¹H-NMR, and Fourier transform infrared spectroscopy were used to obtain the relevant structural information (such as Q3, silanol; Q4, siloxane functional groups of SiO₂NPs; resonance shifts and bending vibrations of PEI25k, –CH₂–CH₂–NH–; and PEG5k, –CH₂–CH₂–O–) from copolymer nanoparticle. Stable complexation of siRNA and nanocomposite particle (wt.%:wt.%) was achieved from 1:5 to 1:15 ratio. Nanocomposite particle (N/P) ratio and siRNA concentration determine the stability and knockdown efficiency of the PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes. It was shown that highly positively charged (zeta potential, +66 mV) PEI25k-PEG5k-graft-SiO₂NPs result in strong affinity with negatively charged siRNA. Confocal microscopy showed intensified cellular uptake of siRNA into cytoplasm of A549 cancer cell utilized for in vitro study. In conclusion, the coherence, graft density of copolymer-SiO₂NPs, and siRNA concentration were found to strongly influence the stability, and hence determine the knockdown efficiency, of PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes.     

Integrated Hollow Mesoporous Silica Nanoparticles for Target Drug/siRNA Co‐Delivery

A hollow mesoporous silica nanoparticle (HMSNP) based drug/siRNA co‐delivery system was designed and fabricated, aiming at overcoming multidrug resistance (MDR) in cancer cells for targeted cancer therapy. The as‐prepared HMSNPs have perpendicular nanochannels connecting to the internal hollow cores, thereby facilitating drug loading and release. The extra volume of the hollow core enhances the drug loading capacity by two folds as compared with conventional mesoporous silica nanoparticles (MSNPs). Folic acid conjugated polyethyleneimine (PEI‐FA) was coated on the HMSNP surfaces under neutral conditions through electrostatic interactions between the partially charged amino groups of PEI‐FA and the phosphate groups on the HMSNP surfaces, blocking the mesopores and preventing the loaded drugs from leakage. Folic acid acts as the targeting ligand that enables the co‐delivery system to selectively bind with and enter into the target cancer cells. PEI‐FA‐coated HMSNPs show enhanced siRNA binding capability on account of electrostatic interactions between the amino groups of PEI‐FA and siRNA, as compared with that of MSNPs. The electrostatic interactions provide the feasibility of pH‐controlled release. In vitro pH‐responsive drug/siRNA co‐delivery experiments were conducted on HeLa cell lines with high folic acid receptor expression and MCF‐7 cell lines with low folic acid receptor expression for comparison, showing effective target delivery to the HeLa cells through folic acid receptor meditated cellular endocytosis. The pH‐responsive intracellular drug/siRNA release greatly minimizes the prerelease and possible side effects of the delivery system. By simultaneously delivering both doxorubicin (Dox) and siRNA against the Bcl‐2 protein into the HeLa cells, the expression of the anti‐apoptotic protein Bcl‐2 was successfully suppressed, leading to an enhanced therapeutic efficacy. Thus, the present multifunctional nanoparticles show promising potentials for controlled and targeted drug and gene co‐delivery in cancer treatment.     

壳聚糖及其衍生物递送miRNA/siRNA的研究进展

微小RNA(microRNA,miRNA)和短链干扰RNA (small interfering RNA,siRNA)是两类具有调节基因表达功能的内源性非编码性小RNA分子.它们已成为多种疾病的潜在治疗药物,逐渐被应用于基因治疗中,而将小RNA应用于基因治疗亟需一种安全高效的递送载体.壳聚糖及其衍生物作为一种可降解、低...     

Synthesis of polyethylenimine grafted with copolymers of polyethylene glycol and polycaprolactone and its potential for siRNA delivery

Copolymers mPEG-PCL were prepared and grafted onto polyethylenimine(PEI) to synthesize copolymers mPEG-PCL-g-PEI with low cytotoxicity.The mPEG-PCL-g-PEI could condense siRNA to form nanoparticles with positive zeta potential.These nanoparticles could delivery siRNA into cells to effectively inhibit the expression of target gene,which suggested that mPEG-PCL-g -PEI could serve as a highly efficient vector for siRNA delivery.     

A Multifunctional Peptide‐Conjugated AIEgen for Efficient and Sequential Targeted Gene Delivery into the Nucleus

Gene therapy has immense potential as a therapeutic approach to serious diseases. However, efficient delivery and real‐time tracking of gene therapeutic agents have not been solved well for successful gene‐based therapeutics. Herein we present a versatile gene‐delivery strategy for efficient and visualized delivery of therapeutic genes into the targeted nucleus. We developed an integrin‐targeted, cell‐permeable, and nucleocytoplasmic trafficking peptide‐conjugated AIEgen named T_DNCP for the efficient and sequential targeted delivery of an antisense single‐stranded DNA oligonucleotide (ASO) and tracking of the delivery process into the nucleus. As compared with T_DNCP/siRNA‐NPs (siRNA functions mainly in the cytoplasm), T_DNCP/ASO‐NPs (ASO functions mainly in the nucleus) exhibited a better interference effect, which further indicates that T_DNCP is a nucleus‐targeting vector. Moreover, T_DNCP/ASO‐NPs showed a favorable tumor‐suppressive effect in vivo.     

Polymer siRNA conjugates synthesised by controlled radical polymerisation

Oligonucleotide therapeutics such as antisense RNA, micro RNA, mRNA and small interfering RNA have great potential to generate a novel therapeutic portfolio within the pharmaceutical market. The promising outlook of oligonucleotide therapeutics lies in their ability to knockdown genes responsible for disease progression. However, the efficient delivery of RNA medicines without causing toxicity remains a major challenge. With growing interest in siRNA therapeutics, a number of synthetic polymers have been developed to facilitate efficient in vitro and in vivo delivery. With the advent of controlled radical polymerisation (CRP) techniques – such as RAFT polymerisation and ATRP – new families of well-defined polymers with narrow molecular weight distribution and predictable molecular architecture potentially suitable to generate siRNA delivery devices are becoming available. In this review article we will describe and discuss how CRP can be utilised to generate siRNA delivery nanodevices.     

Ultra‐pH‐Responsive and Tumor‐Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti‐Cancer Efficacy

RNA interference (RNAi) gene silencing technologies have shown significant potential for treating various diseases, including cancer. However, clinical success in cancer therapy remains elusive, mainly owing to suboptimal in vivo delivery of RNAi therapeutics such as small interference RNA (siRNA) to tumors. Herein, we developed a library of polymers that respond to a narrow pH change (ultra‐pH‐responsive), and demonstrated the utility of these materials in targeted and deep tumor‐penetrating nanoparticle (NP) for in vivo RNAi. The new NP platform is mainly composed of the following key components: i) internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration; ii) polyethylene glycol (PEG) chains to prolong blood circulation; and iii) sharp pH‐responsive hydrophobic polymer to improve endosome escape. Through systematic studies of structure–function relationship, the optimized RNAi NPs (<70 nm) showed efficient gene silencing and significant inhibition of tumor growth with negligible toxicities in vivo.     

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI–PCL) (800 Da–40 kDa) and PEI–PCL–PEI (5–5–5 kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG–PCL) (5 kDa-4 kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100 nm. PEG–PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI–PCL–PEI and PEG–PCL show higher siRNA encapsulation efficiency than PEI–PCL/PEG–PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI–PCL–PEI/PEG–PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.     

A Preloaded Amorphous Calcium Carbonate/Doxorubicin@Silica Nanoreactor for pH‐Responsive Delivery of an Anticancer Drug

Biomedical applications of nontoxic amorphous calcium carbonate (ACC) nanoparticles have mainly been restricted because of their aqueous instability. To improve their stability in physiological environments while retaining their pH‐responsiveness, a novel nanoreactor of ACC–doxorubicin (DOX)@silica was developed for drug delivery for use in cancer therapy. As a result of its rationally engineered structure, this nanoreactor maintains a low drug leakage in physiological and lysosomal/endosomal environments, and responds specifically to pH 6.5 to release the drug. This unique ACC–DOX@silica nanoreactor releases DOX precisely in the weakly acidic microenvironment of cancer cells and results in efficient cell death, thus showing its great potential as a desirable chemotherapeutic nanosystem for cancer therapy.     

A novel polyethyleneimine-decorated FeOOH nanoparticle for efficient siRNA delivery

Despite the promising prospect of small interfering RNA(siRNA) for the treatment of diverse diseases,it remains challenging to develop novel delive ry materials to desired tissues and cells.In this study,a novel iron oxyhydroxide(FeOOH) nanoparticle(NP) whose surface was modified with branched polyetherimide(PEI) was developed to deliver siRNA into the cancer cells.It was demonstrated that PEI-FeOOH(PFeOOH) efficiently complexed siRNA,mediated effective cellular uptake and endosomal escape,thereby triggering robust gene silencing in vitro.In addition,PFeOOH/siRNA formulation loading with anti-RRM2 siRNA effectively inhibited the growth of tumor tissues,and exhibited excellent safety profiles in vivo.Therefore,this study conceptually provided a FeOOH-based nucleic acid delivery vesicle which can potentially use to achieve diagnosis and therapy simultaneously.     

General Route to Multifunctional Uniform Yolk/Mesoporous Silica Shell Nanocapsules: A Platform for Simultaneous Cancer‐Targeted Imaging and Magnetically Guided Drug Delivery

Hollow mesoporous SiO₂ (mSiO₂) nanostructures with movable nanoparticles (NPs) as cores, so‐called yolk‐shell nanocapsules (NCs), have attracted great research interest. However, a highly efficient, simple and general way to produce yolk‐mSiO₂ shell NCs with tunable functional cores and shell compositions is still a great challenge. A facile, general and reproducible strategy has been developed for fabricating discrete, monodisperse and highly uniform yolk‐shell NCs under mild conditions, composed of mSiO₂ shells and diverse functional NP cores with different compositions and shapes. These NPs can be Fe₃O₄ NPs, gold nanorods (GNRs), and rare‐earth upconversion NRs, endowing the yolk‐mSiO₂ shell NCs with magnetic, plasmonic, and upconversion fluorescent properties. In addition, multifunctional yolk‐shell NCs with tunable interior hollow spaces and mSiO₂ shell thickness can be precisely controlled. More importantly, fluorescent‐magnetic‐biotargeting multifunctional polyethyleneimine (PEI)‐modified fluorescent Fe₃O₄@mSiO₂ yolk‐shell nanobioprobes as an example for simultaneous targeted fluorescence imaging and magnetically guided drug delivery to liver cancer cells is also demonstrated. This synthetic approach can be easily extended to the fabrication of multifunctional yolk@mSiO₂ shell nanostructures that encapsulate various functional movable NP cores, which construct a potential platform for the simultaneous targeted delivery of drug/gene/DNA/siRNA and bio‐imaging.     

Anti-tumor effects of combined doxorubicin and siRNA for pulmonary delivery

Direct administration of drugs and genes to the lungs by pulmonary delivery offers a potential effective therapy for lung cancers.In this study,combined doxorubicin(DOX)and Bcl2 siRNA was employed for cancer therapy using polyethylenimine(PEI)as the carrier of Bcl2 siRNA.Most of the DOX and siRNA possessed high cellular uptake efficiency in B16F10 cells,which was proved by FCM and CLSM analysis. Real-time PCR showed that PEI/Bcl2 siRNA exhibited high gene silencing efficiency with 70% Bcl2 mRNA being knocked down.The combination of DOX and siRNA could enhance the cell proliferation inhibition and the cell apoptosis against B16F10 cells compared to free DOX or PEI/Bcl2 siRNA.Furthermore,the biodistribution of DOX and siRNA via pulmonary administration was studied in mice with B16F10 metastatic lung cancer.The results showed that most of the DOX and siRNA were accumulated in lungs and lasted at least for 3 days,which suggested that combined DOX and siRNA by pulmonary administration may have high anti-tumor effects for metastatic lung cancer treatment in vivo.     

Polyethyleneimine/poly-(γ-glutamic acid)/poly(lactide-co-glycolide) nanoparticles for loading and releasing antiretroviral drug

Nanoparticles (NPs) with ternary components of polyethyleneimine (PEI), poly-(γ-glutamic acid) (γ-PGA), and poly(lactide-co-glycolide) (PLGA) were applied to carry and release saquinavir (SQV). Hydrophobic SQV was encapsulated in the particle core composed of PLGA to form SQV-PLGA NPs, and the surface of SQV-PLGA NPs was grafted successively with hydrophilic γ-PGA and PEI (PEI/γ-PGA/SQV-PLGA NPs). The morphological images revealed that PEI/γ-PGA/SQV-PLGA NPs were spheroid-like, in general. An increase in the concentration of didecyl dimethylammonium bromide and a reduction in the dose of SQV enhanced the entrapment efficiency of SQV in PLGA NPs. In addition, an increment in the molecular weight of γ-PGA reduced the grafting efficiency of PEI on γ-PGA/SQV-PLGA NPs. An increase in the weight percentage of PEI enhanced the average particle diameter. However, the grafting efficiency of PEI on γ-PGA/SQV-PLGA NPs and the dissolution rate of SQV from PEI/γ-PGA/SQV-PLGA NPs reduced when the weight percentage of PEI increased. PEI/γ-PGA/SQV-PLGA NPs are an innovative drug delivery system and can be used for antiretroviral trials.     

疏水高分子介导可动芯SiO2杂化纳米粒子的仿生合成

在疏水高分子胶体模板——含氟丙烯酸酯(FA)共聚物乳胶粒中引入能够介导SiO2原位沉积的聚胺催化活性点-甲基丙烯酰氧乙基三甲基氯化铵(DMC),以四甲氧基硅烷(TMOS)为硅源,在环境条件下可控合成了核壳型FA共聚物/SiO2杂化纳米粒子.高温煅烧除去聚合物核质,可得到中空的SiO2纳米粒子,结合FTIR、EDX、TGA以及XPS等表征数据印证了SiO2的沉积主要发生在聚合物模板的表面.进一步考察了反应条件,如聚胺功能单体DMC的浓度、TMOS的浓度以及反应时间对SiO2杂化纳米粒子的形貌与组成的影响.实验结果表明增加DMC或者TMOS的浓度,适当延长反应时间,均可增加SiO2粒子的沉积速率,导致SiO2壳层的厚度增加,并且杂化粒子的形貌由凹陷多褶皱的核壳结构向可动芯结构转变.由于FA共聚物模板的强疏水性,增加有机核层和无机壳层间的不相容排斥,最终导致核壳层间空腔的形成,得到含可动芯的核壳型SiO2杂化粒子.     

Polyethylenimine‐assisted Synthesis of Au Nanoparticles for Efficient Syngas Production

The ability to capture, store, and use CO₂ is important for remediating greenhouse‐gas emissions and combatting global warming. Herein, Au nanoparticles (Au‐NPs) are synthesized for effective electrochemical CO₂ reduction and syngas production, using polyethylenimine (PEI) as a ligand molecule. The PEI‐assisted synthesis provides uniformly sized 3‐nm Au NPs, whereas larger irregularly shaped NPs are formed in the absence of PEI in the synthesis solution. The Au‐NPs synthesized with PEI (PEI?Au/C, average PEI Mw=2000) exhibit improved CO₂ reduction activities compared to Au‐NPs formed in the absence of PEI (bare Au NPs/C). PEI?Au/C displays a 34 % higher activity toward CO₂ reduction than bare Au NPs/C; for example, PEI?Au/C exhibits a CO partial current density (j_CO) of 28.6 mA cm^?2 at ?1.13 V_RHE, while the value for bare Au NPs/C is 21.7 mA cm^?2; the enhanced j_CO is mainly due to the larger surface area of PEI?Au/C. Furthermore, the PEI?Au/C electrode exhibits stable performance over 64 h, with an hourly current degradation rate of 0.25 %. The developed PEI?Au/C is employed in a CO₂‐reduction device coupled with an IrO₂ water‐oxidation catalyst and a proton‐conducting perfluorinated membrane to form a PEI?Au/C|Nafion|IrO₂ membrane‐electrode assembly. The device using PEI?Au/C as the CO₂‐reduction catalyst exhibits a j_CO of 4.47 mA/cm² at 2.0 V_cell. Importantly, the resulted PEI?Au/C is appropriate for efficient syngas production with a CO ratio of around 30–50 %.     

Systemic Delivery of Bc12‐Targeting siRNA by DNA Nanoparticles Suppresses Cancer Cell Growth

Short interfering RNA (siRNA) is a promising molecular tool for cancer therapy, but its clinical success is limited by the lack of robust in vivo delivery systems. Rationally designed DNA nanoparticles (DNPs) have emerged as facile delivery vehicles because their physicochemical properties can be precisely controlled. Nonetheless, few studies have used DNPs to deliver siRNAs in vivo, and none has demonstrated therapeutic efficacy. Herein, we constructed a number of DNPs of rectangular and tubular shapes with varied dimensions using the modular DNA brick method for the systemic delivery of siRNA that targets anti‐apoptotic protein Bcl2. The siRNA delivered by the DNPs inhibited cell growth both in vitro and in vivo, which suppressed tumor growth in a xenograft model that specifically correlated with Bcl2 depletion. This study suggests that DNPs are effective tools for the systemic delivery of therapeutic siRNA and have great potential for further clinical translation.     

MicroRNA Delivery with Bioreducible Polyethylenimine as a Non‐Viral Vector for Breast Cancer Gene Therapy

Polyethylenimines (PEIs) are outstanding macromolecules belonging to the polycations used in gene transfection. The transfection efficiency and cytotoxicity of PEIs increase with the increase in their molecular weight. To break up the correlation between transfection efficiency and cytotoxicity for non‐viral gene delivery, disulfide cross‐linked polyethylenimine (PEI‐SS) has been widely employed as highly efficient gene vectors for DNA/siRNA delivery in numerous efforts. In this work, PEI‐SS is described as a non‐viral vector for miRNA delivery for the first time. PEI‐SS is synthesized via cross‐linking using disulfide bonds as the cross‐linker from low molecular weight PEI. PEI‐SS can efficiently bind anti‐miR‐155 to form the polyplex with nano‐sized spherical structures in the size range of 10–100 nm. The polyplex is degraded by glutathione (GSH, a reducing agent) in cancer cells. Anti‐miR‐155 is then released to efficiently inhibit tumor growth.     

Unzipping and binding of small interfering RNA with single walled carbon nanotube: a platform for small interfering RNA delivery

In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference.