Anti-EpCAM functionalized graphene oxide vector for tumor targeted siRNA delivery and cancer therapy

Graphene oxide(GO) has emerged as a potential drug delivery vector. For siRNA delivery, GO should be modified to endow it with gene delivery ability and targeting effect. However, the cationic materials used previously usually had greater toxicity. In this study, GO was modified with a non-toxicity cationic material(chitosan) and a tumor specific monoclonal antibody(anti-EpCAM) for the delivery of survivin-siRNA(GCE/siRNA). And the vector(GCE) prepared was proved with excellent biosafety and tumor targeting effect. The GCE exhibited superior performance in loading si RNA, maintained stability in different solutions and showed excellent protection effect for survivin-siRNA in vitro. The gene silencing results in vitro showed that the m RNA level and protein level were down-regulated by 48.24% ± 2.50% and 44.12% ± 3.03%, respectively, which was equal with positive control( P > 0.05). It was also demonstrated that GCE/siRNA had a strong antitumor effect in vitro, which was attributed to the efficient antiproliferation, and migration and invasion inhibition effect of GCE/siRNA. The results in vivo indicated that GCE could accumulate siRNA in tumor tissues. The tumor inhibition rate of GCE/siRNA 54.74% ± 5.51% was significantly higher than control 4.87% ±8.49%. Moreover, GCE/siRNA showed no toxicity for blood and main organs, suggesting that it is a biosafety carrier for gene delivery. Taken together, this study provides a novel design strategy for gene delivery system and siRNA formulation.

Characterization of modified mesoporous silica nanoparticles as vectors for siRNA delivery

Gene therapy using siRNA molecules is nowadays considered as a promising approach. For successful therapy, development of a stable and reliable vector for siRNA is crucial. Non-viral and non-organic vectors like mesoporous silica nanoparticles(MSN) are associated with lack of most viral vector drawbacks, such as toxicity, immunogenicity, but also generally a low nucleic acid carrying capacity. To overcome this hurdle, we here modified the pore walls of MSNs with surface-hyperbranching polymerized poly(ethyleneimine)(hbPEI), which provides an abundance of amino-groups for loading of a larger amount of siRNA molecules via electrostatic adsorption. After loading, the particles were covered with a second layer of pre-polymerized PEI to provide better protection of siRNA inside the pores, more effective cellular uptake and endosomal escape. To test the transfection efficiency of PEI covered si RNA/MSNs, MDA-MB 231 breast cancer cells stably expressing GFP were used. We demonstrate that PEI-coated si RNA/MSN complexes provide more effective delivery of si RNAs compared to unmodified MSNs. Thus, it can be concluded that appropriately surface-modified MSNs can be considered as prospective vectors for therapeutic siRNA delivery.

Preparation and Characterization of pH-Responsive Charge Reversal Nanocomposite for miRNA Delivery

pH-responsive charge reversal loaded miRNA nanocomposite was prepared by electrostatic self-assembly.The morphology,particle size and zeta potential of the nanocomposites were analyzed by transmission electron microscopy and dynamic light scattering.The synthesis of the polymer was analyzed by^(1)H-NMR.The zeta-potential changes and cellular uptake effects of the nanocomplexes under different pH environments were investigated.The experimental results show that the surface morphology of the nanocomposite is spherical,and the average particle size is about 135 nm.As the pH value of the solution gradually decreases,the surface charge of the nanocomposite reverses from negative charge to positive charge(from-9.4 to+17.1 mV).Cellular uptake mediated by pH-responsive nanocomposite is selective for tumor cells,and the cellular uptake effect in tumor cells at pH 6.5 was approximately 3 times higher than that at pH 7.4.This pH responsive charge reversal nanocomposite has promising application prospects for gene delivery in the weak acid environment of tumors.

Self-crosslinkable chitosan-hyaluronic acid dialdehyde nanoparticles for CD44-targeted siRNA delivery to treat bladder cancer

Bladder cancer is one of the concerning malignancies worldwide,which is lacking effective targeted therapy.Gene therapy is a potential approach for bladder cancer treatment.While,a safe and effective targeted gene delivery system is urgently needed for prompting the bladder cancer treatment in vivo.In this study,we confirmed that the bladder cancer had CD44 overexpression and small interfering RNAs(siRNA)with high interfere to Bcl2 oncogene were designed and screened.Then hyaluronic acid dialdehyde(HAD)was prepared in an ethanol-water mixture and covalently conjugated to the chitosan nanoparticles(CS-HAD NPs)to achieve CD44 targeted siRNA delivery.The in vitro and in vivo evaluations indicated that the siRNA-loaded CS-HAD NPs(siRNA@CS-HAD NPs)were approximately 100 nm in size,with improved stability,high siRNA encapsulation efficiency and low cytotoxicity.CS-HAD NPs could target to CD44 receptor and deliver the therapeutic siRNA into T24 bladder cancer cells through a ligand-receptor-mediated targeting mechanism and had a specific accumulation capacity in vivo to interfere the targeted oncogene Bcl2 in bladder cancer.Overall,a CD44 targeted gene delivery system based on natural macromolecules was developed for effective bladder cancer treatment,which could be more conducive to clinical application due to its simple preparation and high biological safety.

Injectable self-healing hydrogel with siRNA delivery property for sustained STING silencing and enhanced therapy of intervertebral disc degeneration

Inflammatory responses of nucleus pulposus(NP)can induce imbalanced anabolism and catabolism of extracellular matrix,and the cytosolic dsDNA accumulation and STING-NF-κB pathway activation found in NP inflammation are considered as fairly important cause of intervertebral disc(IVD)degeneration.Herein,we constructed a siSTING delivery hydrogel of aldehyde hyaluronic acid(HA-CHO)and poly(amidoamine)PAMAM/siRNA complex to intervene the abnormal STING signal for IVD degeneration treatment,where the formation of dynamic Schiff base bonds in the system(siSTING@HPgel)was able to overcome the shortcomings such as low cellular uptake,short half-life,and rapid degradation of siRNA-based strategy.PAMAM not only formed complexes with siRNA to promote siRNA transfection,but also served as dynamic crosslinker to construct hydrogel,and the injectable and self-healing hydrogel efficiently and steadily silenced STING expression in NP cells.Finally,the siSTING@HPgel significantly eased IVD inflammation and slowed IVD degeneration by prolonging STING knockdown in puncture-induced IVD degeneration rat model,revealing that STING pathway was a therapeutic target for IVD degeneration and such novel hydrogel had great potential for being applied to many other diseases for gene delivery.

An ionizable supramolecular dendrimer nanosystem for effective siRNA delivery with a favorable safety profile

Gene therapy using small interfering RNA(siRNA)is emerging as a novel therapeutic approach to treat various diseases.However,safe and efficient siRNA delivery still constitutes the major obstacle for clinical implementation of siRNA therapeutics.Here we report an ionizable supramolecular dendrimer vector,formed via self-assembly of a small amphiphilic dendrimer,as an effective siRNA delivery system with a favorable safety profile.By virtue of the ionizable tertiary amine terminals,the supramolecular dendrimer has a low positively charged surface potential and no notable cytotoxicity at physiological pH.Nonetheless,this ionizable feature imparted sufficient surface charge to the supramolecular dendrimer to enable formation of a stable complex with siRNA via electrostatic interactions.The resulting siRNA/dendrimer delivery system had a surface charge that was neither neutral,thus avoiding aggregation,nor too high,thus avoiding cytotoxicity,but was sufficient for favorable cellular uptake and endosomal release of the siRNA.When tested in different cancer cell lines and patient-derived cancer organoids,this dendrimer-mediated siRNA delivery system effectively silenced the oncogenes Myc and Akt2 with a potent antiproliferative effect,outperforming the gold standard vector,Lipofectamine 2000.Therefore,this ionizable supramolecular dendrimer represents a promising vector for siRNA delivery.The concept of supramolecular dendrimer nanovectors via self-assembly is new,yet easy to implement in practice,offering a new perspective for supramolecular chemistry in biomedical applications.

Bone site-specific delivery of siRNA

Small interfering RNAs （siRNA） have enormous potential as therapeutics to target and treat various bone disor- ders such as osteoporosis and cancer bone metastases. However, effective and specific delivery of siRNA therapeu- tics to bone and bone-specific cells in vivo is very challenging. To realize the full therapeutic potential of siRNA in treating bone disorders, a safe and efficient, tissue- and cell-specific delivery system must be developed. This review focuses on recent advances in bone site-specific delivery of siRNA at the tissue or cellular level. Bone-targeted nanoparticulate siRNA carriers and various bone-targeted moieties such as bisphosphonates, oligopeptides （Asp）8 and （AspSerSer）6, and aptamers are highlighted. Incorporation of these bone-seeking targeting moieties into siRNA carriers allows for recognition of different sub-tissue functional domains of bone and also specific cell types residing in bone tissue. It also provides a means for bone-formation surface-, bone-resorption surface-, or osteoblast- specific targeting and transportation of siRNA therapeutics. The discussion mainly focuses on systemic and local bone-specific delivery of siRNA in osteoporosis and bone metastasis preclinical models.

Natural Polyphenol Inspired Polycatechols for Efficient siRNA Delivery

There is a continuing quest to rationally fabricate polymeric biomaterials with both high transfection efficiency and minimal toxicity for the emerging opportunities in small interfering RNA(siRNA)delivery.Recently,this goal was promoted highly by developing a robust and efficient strategy to facilitate polymer-mediated RNAi using natural polyphenols with multiple phenol groups that could condense siRNA effectively into negatively charged nanoparticles(NPs).Further coating of these NPs with cationic polymers of low molecular weight enabled their intracellular siRNA delivery.Inspired by the structural and functional features of natural polyphenols,we aimed to further the development of low molecular weight polycatechols as a new class of efficient and biocompatible polymers for siRNA delivery in our current study.The fabricated polycatechols have benefits of requiring only one-step fabrication toward efficient siRNA nanoformulations.Moreover,they could deliver siRNA into cells and silence target genes both in vitro and in vivo.The resulting polycatechol/siRNA formulations were also functionally competent,demonstrating a successful,profound downregulation of a proinflammatory enzyme to attenuate chronic intestinal inflammation in an intestinal injury model.This study provides a new approach in chemistry for the development of efficient synthetic polymers for therapeutic siRNA delivery.

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.

Novel cationic lipid with reduction-responsive cleavable hydrophobic tail for siRNA delivery

To achieve a higher transfection efficiency and lower toxicity, a novel herringbone-like cationic lipid（2 ss HLL） composed of hydrophilic aspartic acid linked with two reduction-responsive cleavable hydrophobic oleic acid tails was synthesized and assessed in this study. In our results, the cationic nanoplexes with a uniform spherical shape and a particle size of ~150 nm were successfully prepared by the electrostatic interaction between si RNAs and 2 ss HLL-based liposomes. From the results evaluated in Hep G2 cells, it was shown that the nanoplexes exhibited high cellular uptake of si RNA with a low cytotoxicity. Moreover, the significant down-regulation effects of 2 ss HLL/si EGFR nanoplexes on target m RNA were displayed by RT-PCR analysis, which were similar to those of Lipofectamine2000. It suggested that the enhanced si RNA gene silencing efficiency was probably attributed to the detachment of hydrophobic tail chains induced by reduction-responsive cleavage. This mechanism was also confirmed by the changes of size distribution and si RNA release of nanoplexes in the reductive environment and DTT-absence condition. Overall, we believed that the redox-active herringbone-like 2 ss HLL would be a potential nanocarrier towards si RNA delivery.

Remodeling the tumor immune microenvironment via siRNA therapy for precision cancer treatment

How to effectively transform the pro-oncogenic tumor microenvironments(TME)surrounding a tumor into an anti-tumoral never fails to attract people to study.Small interfering RNA(siRNA)is considered one of the most noteworthy research directions that can regulate gene expression following a process known as RNA interference(RNAi).The research about siRNA delivery targeting tumor cells and TME has been on the rise in recent years.Using siRNA drugs to silence critical proteins in TME was one of the most efficient solutions.However,the manufacture of a siRNA delivery system faces three major obstacles,i.e.,appropriate cargo protection,accurately targeted delivery,and site-specific cargo release.In the following review,we summarized the pharmacological actions of siRNA drugs in remolding TME.In addition,the delivery strategies of siRNA drugs and combination therapy with siRNA drugs to remodel TME are thoroughly discussed.In the meanwhile,the most recent advancements in the development of all clinically investigated and commercialized siRNA delivery technologies are also presented.Ultimately,we propose that nanoparticle drug delivery siRNA may be the future research focus of oncogene therapy.This summary offers a thorough analysis and roadmap for general readers working in the field.

Dual-targeted lung cancer therapy via inhalation delivery of UCNP-siRNA-AS1411 nanocages

Objective:Although great progress has been made in the field of siRNA gene therapy,safe,efficient,and targeted delivery of siRNA are still major challenges in siRNA therapeutics.Methods:We developed an up-conversion nanoparticle-based nanocage system.This system protected the siRNA from being degraded by nucleases in organisms and selectively delivered the siRNAs to the tumor sites,due to modifications of targeted molecules on the surfaces of nanocages and local inhalation.Results:The siRNAs delivered by the up-conversion nanoparticle nanocages were protected from degradation in transit to the tumor sites,where they accumulated.Compared with the passive target and control groups,the up-conversion nanoparticles based on the nanocage system showed a tumor suppressive effect after approximately 3 weeks of treatment.Conclusions:The up-conversion nanoparticle nanocages efficiently delivered vascular endothelial growth factor siRNAs to tumor sites.Mice with lung tumors treated with tumors targeting up-conversion nanoparticle nanocages showed steady body weight changes,high tumor inhibition ratios,and longer survival times.

Construction of Magnetic Resonance Imaging Visible Polymeric Vector for Efficient Tumor Targeted siRNA Delivery

RNA interference(RNAi),known for the highly efficient targeted gene silencing,has been demonstrated to be a promising means for cancer treatment.Meanwhile,an effective approach for siRNA delivery is urgently needed to meet the needs for its clinical application.Herein,we constructed a polymeric vector labeled with superparamagnetic iron oxide(SPIO)for magnetic resonance imaging(MRI)visible siRNA delivery.EGFR antibody was also modified to the surface of nanodrug to enhance the delivery effect.Our results showed that the vector exhibited great siRNA complexation ability and mediated an increased endocytosis of siRNA without obvious cytotoxicity.Besides,both in vitro and in vivo studies evidenced the vector could effectively deliver siRNA into tumor cells,exert highly interfering effect,and show potent MR imaging capacity.The study provides a promising MRI-visible and EGFR targeting delivery system to improve RNAi efficacy for cancer therapy.

Supramolecular Assembly Models of siRNA Delivery Systems

Small interfering RNA(siRNA)technology,which could be able to silence specific targeted mRNAs rapidly and efficiently,has been regarded as a novel and potent gene therapeutic approach for diseases,such as cancers,in-fections,inflammations and neurological disorders.However,some conspicuous intrinsic defects of siRNA including the poor cellular uptake,low biological stability,unfavorable pharmacokinetics and bio-distribution properties,have hindered further applications in clinic.Therefore,it is prerequisite to design safe and effective siRNA delivery systems for the successful development of siRNA-based various diseases therapeutics.This review aims to analyze and summarize these supramolecular assembly models in the process of siRNA delivery,namely recent nanotech-nological strategies for carrying siRNA by using diverse non-viral vehicles such as cationic liposomes,stable nucleic acid lipid particles(SNALPs),cationic polymers,cationic dendrimers,inorganic nanoparticles and others.Eventually,it is concluded with future perspectives of siRNA therapeutics in the field of non-viral carriers.

A Combinatorial Approach Based on Nucleic Acid Assembly and Electrostatic Compression for siRNA Delivery

A combinatorial strategy based on nucleic acid assembly and electrostatic complexation is developed for efficient small interfering ribonucleic acid(siRNA)delivery.In this approach,siRNAs are first loaded into a well-defined nanotube through programmable nucleic acid self-assembly.Compared to small rigid siRNA duplex,the obtained siRNA-bearing nanotube with large architecture is more readily to complex with cationic and ionizable poly(β-amino ester),resulting in the formation of a novel platform for efficient siRNA delivery.

Collaborative assembly-mediated siRNA delivery for relieving inflammation-induced insulin resistance

Obesity plays a primary causative role in insulin resistance and hyperglycemia that contributes to type 2 diabetes.Excess lipid storage in the liver renders activation of the resident macrophages and chronic secretion of inflammatory mediators,therefore causing or aggravating insulin resistance.Herein,we develop collaborative assemblies using a“one-pot”synthesis method for macrophage-specific delivery of small interfering RNAs(siRNAs)that target the inflammatory proteins.Ternary nanocomplex(NC)composed of the siRNA molecule,a synthetic thiol-bearing methacrylated hyaluronic acid(sm-HA)and protamine forms through an electrostatic-driven physical assembly,which is chemically crosslinked to acquire the collaboratively assembled nanocapsule(cNC)concurrently.The obtained cNC displays significantly higher stability than NC.Functional moieties as flexible assembly units can be easily equipped on cNC for long circulation,active targeting,or controlled siRNA release.cNC-F decorated with folic acid,a macrophage-targeting ligand promotes the siRNA accumulation in the activated macrophages in the liver of the obese mouse model.cNC-F loaded with siRNA targeting inflammatory indicators efficiently control the macrophage inflammatory response by reducing the expression of the inflammatory proteins(>40%reduction)and ameliorating the insulin resistance symptoms of the obese mice.

Elaborately engineering of lipid nanoparticle for targeting delivery of siRNA and suppressing acute liver injury

Small interfering RNA(siRNA)-based gene silencing has been considered as a potential therapy modality against inflammatory diseases.Nevertheless,the effective delivery of siRNA to desired destination still remains challenging due to poor stability,high molecular weight and negative charge.Currently,ionizable lipid nanoparticle(LNP)has been extensively used as vector for effective delivery of siRNA.Herein,we report a mannose-modified LNP(M-MC_(3) LNP@TNFα)loading tumor necrosis factorα(TNFα)siRNA for targeting liver macrophages,achieving effectively inhibit acute liver injury.The M-MC_(3) LNP@TNFαnot only increases the internalization of LNP by macrophages,but also enhances the gene silencing efficiency of TNFαin vitro.Additionally,the M-MC_(3) LNP@TNFαexhibits higher accumulation in liver of healthy mice than that of MC_(3) LNP@TNFα(un-modified LNP)owing to the targeting effect of mannose.As expected,the M-MC_(3) LNP@TNFαsignificantly suppresses the expression of TNFαand ameliorates liver damage in acute liver injury model.Such a LNP targeting siRNA delivery holds great potential for the treatment of diseases associated with liver in the future.

Engineering Cell-Selective Charge-Altering Lipid Nanoparticles for Efficient siRNA Delivery in Vivo

Lipid nanoparticles(LNPs)have emerged as a powerful platform for RNA delivery;the chemical engineering of LNP for efficient delivery of RNA into cytosol remains critical but challenging.One promising strategy is the use of permanently positively charged lipids,which have been shown to enhance the stability and delivery efficiency of LNPs.However,the resulting strong electrostatic interactions reduced the RNA release capacity from the lipoplexes.Herein,we engineered a hydrogen peroxide(H_(2)O_(2))-triggered charge-altering LNP(CALNP)for efficient small interfering RNA(siRNA)delivery and tumor therapy in mice.The incorporation of phenylboronic acid(PBA)into ionizable lipids generated permanently positively charged lipids.A CALNP with optimal lipid formulations was identified,exhibiting enhanced transfection efficiency with effective lysosomal escape through dual effects of electrostatic interaction and ligand-receptor binding.H_(2)O_(2)-triggered removal of PBA groups regenerated ionizable LNP with reduced positive charges at physiological pH,allowing cell-selective siRNA release in the cytoplasm.Our results demonstrated that CALNPs exhibited improved siRNA transfection and gene silencing efficiency.We also showed potent CALNP activity against the polo-like kinase 1(Plk1)gene by effectively silencing Plk1 mRNA and subsequent suppression of tumor growth.Collectively,these findings highlighted the potential of CALNP as an efficient platform for RNA delivery and tumor therapeutics.

MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

Posttraumatic osteoarthritis(PTOA)patients are often diagnosed by X-ray imaging at a middle-late stage when drug interventions are less effective.Early PTOA is characterized by overexpressed matrix metalloprotease 13(MMP13).Herein,we constructed an integrated diagnosis and treatment micelle modified with MMP13 enzyme-detachable,cyanine 5(Cy5)-containing PEG,black hole quencher-3(BHQ3),and cRGD ligands and loaded with siRNA silencing MMP13(siM13),namely ERMs@siM13.ERMs@siM13 could be cleaved by MMP13 in the diseased cartilage tissues to detach the PEG shell,causing cRGD exposure.Accordingly,the ligand exposure promoted micelle uptake by the diseased chondrocytes by binding to cell surfaceαvβ3 integrin,increasing intracellular siM13 delivery for on-demand MMP13 downregulation.Meanwhile,the Cy5 fluorescence was restored by detaching from the BHQ3-containing micelle,precisely reflecting the diseased cartilage state.In particular,the intensity of Cy5 fluorescence generated by ERMs@siM13 that hinged on the MMP13 levels could reflect the PTOA severity,enabling the physicians to adjust the therapeutic regimen.Finally,in the murine PTOA model,ERMs@siM13 could diagnose the early-stage PTOA,perform timely interventions,and monitor the OA progression level during treatment through a real-time detection of MMP13.Therefore,ERMs@siM13 represents an appealing approach for early-stage PTOA theranostics.

Tumor-targeted delivery of siRNA to silence Sox2 gene expression enhances therapeutic response in hepatocellular carcinoma

RNA interference(RNAi)is one of the most promising methods for the treatment of malignant tumors.However,developing an efficient biocompatible delivery vector for small interfering RNA(siRNA)remains a challenging issue.This study aimed to prepare a non-viral tumor-targeted carrier,named RGDfC-modified functionalized selenium nanoparticles(RGDfC-SeNPs).RGDfC-SeNPs were used to selectively deliver siSox2 to HepG2 liver cancer cells and tissues for the treatment of hepatocellular carcinoma(HCC).In the current study,RGDfC-SeNPs were successfully synthesized and characterized.It was shown that RGDfC-SeNPs could effectively load siSox2 to prepare an antitumor prodrug RGDfC-Se@siSox2.RGDfC-Se@siSox2 exhibited selective uptake in HepG2 liver cancer cells and LO2 normal liver cells,indicating RGDfC-SeNPs could effectively deliver siSox2 to HepG2 liver cancer cells.RGDfC-Se@siSox2 entered HepG2 cells via clathrin-mediated endocytosis by firstly encircling the cytoplasm and then releasing siSox2 in the lysosomes.RGDfC-Se@siSox2 could effectively silence Sox2 and inhibit the proliferation,migration and invasion of HepG2 cells.RGDfC-Se@siSox2 induced HepG2 cells apoptosis most likely via overproduction of reactive oxygen species and disruption of the mitochondrial membrane potentials.Most importantly,RGDfC-Se@siSox2 significantly inhibited the tumor growth in HepG2 tumor-bearing mice without obvious toxic side effects.These studies indicated that RGDfC-SeNPs may be an ideal gene carrier for delivering siSox2 to HepG2 cells and that RGDfC-Se@siSox2 may be a novel and highly specific gene-targeted prodrug therapy for HCC.