Delivery systems for siRNA drug development in cancer therapy

Since the discovery of the Nobel prize-winning mechanism of RNA interference(RNAi)ten years ago,it has become a promising drug target for the treatment of multiple diseases,including cancer.There have already been some successful applications of siRNA drugs in the treatment of age-related macular degeneration and respiratory syncytial virus infection.However,significant barriers still exist on the road to clinical applications of siRNA drugs,including poor cellular uptake,instability under physiological conditions,off-target effects and possible immunogenicity.The successful application of siRNA for cancer therapy requires the development of clinically suitable,safe and effective drug delivery systems.Herein,we review the design criteria for siRNA delivery systems and potential siRNA drug delivery systems for cancer therapy,including chemical modifications,lipidbased nanovectors,polymer-mediated delivery systems,conjugate delivery systems,and others.

Stimuli-Responsive Gene Delivery Nanocarriers for Cancer Therapy

Gene therapy provides a promising approach in treating cancers with high efficacy and selectivity and few adverse effects.Currently,the development of functional vectors with safety and effectiveness is the intense focus for improving the delivery of nucleic acid drugs for gene therapy.For this purpose,stimuli-responsive nanocarriers displayed strong potential in improving the overall efficiencies of gene therapy and reducing adverse effects via effective protection,prolonged blood circulation,specific tumor accumulation,and controlled release profile of nucleic acid drugs.Besides,synergistic therapy could be achieved when combined with other therapeutic regimens.This review summarizes recent advances in various stimuliresponsive nanocarriers for gene delivery.Particularly,the nanocarriers responding to endogenous stimuli including pH,reactive oxygen species,glutathione,and enzyme,etc.,and exogenous stimuli including light,thermo,ultrasound,magnetic field,etc.,are introduced.Finally,the future challenges and prospects of stimuli-responsive gene delivery nanocarriers toward potential clinical translation are well discussed.The major objective of this review is to present the biomedical potential of stimuli-responsive gene delivery nanocarriers for cancer therapy and provide guidance for developing novel nanoplatforms that are clinically applicable.

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.

Small interfering RNA-based molecular therapy of cancers

RNA interference(RNAi)has become a gold standard for validating gene function in basic life science research and provides a promising therapeutic modality for cancer and other diseases.This minireview focuses on the potential of small interfering RNAs(siRNAs)in anticancer treatment,including the establishment and screening of cancer-associated siRNA libraries and their applications in anticancer drug target discovery and cancer therapy.This article also describes the current delivery approaches of siRNAs using lipids,polymers,and,in particular,gold nanoparticles to induce significant gene silencing and tumor growth regression.

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.

Identification of genes associated with gall bladder cell carcinogenesis:Implications in targeted therapy of gall bladder cancer

Gall bladder cancer(GBC)is becoming a very devastating form of hepatobiliary cancer in India.Every year new cases of GBC are quite high in India.Despite recent advanced multimodality treatment options,the survival of GBC patients is very low.If the disease is diagnosed at the advanced stage(with local nodal metastasis or distant metastasis)or surgical resection is inoperable,the prognosis of those patients is very poor.So,perspectives of targeted therapy are being taken.Targeted therapy includes hormone therapy,proteasome inhibitors,signal transduction and apoptosis inhibitors,angiogenesis inhibitors,and immunotherapeutic agents.One such signal transduction inhibitor is the specific short interfering RNA(siRNA)or short hairpin RNA(shRNA).For developing siRNAmediated therapy shRNA,although several preclinical studies to evaluate the efficacy of these key molecules have been performed using gall bladder cells,many more clinical trials are required.To date,many such genes have been identified.This review will discuss the recently identified genes associated with GBC and those that have implications in its treatment by siRNA or shRNA.

Room for improvement in the treatment of pancreatic cancer: Novel opportunities from gene targeted therapy

Pancreatic cancer is one of the highest and in fact,unchanged mortality-associated tumor,with an exceptionally low survival rate due to its challenging diagnostic approach.So far,its treatment is based on a combination of approaches(such as surgical resection with or rarely without chemotherapeutic agents),but with finite limits.Thus,looking for additional space to improve pancreatic tumorigenesis therapeutic approach,research has focused on gene therapy with unexpectedly growing horizons not only for the treatment of inoperable pancreatic disease,but also for its early stages.In vivo gene delivery viral vectors,despite few disadvantages(possible immunogenicity,toxicity,mutagenicity,or high cost),could be one of the most efficient cancer gene therapeutic strategies for clinical application due to their superiority compared with other systems(ex vivo delivery strategies).Their dominance consists of simple preparation,easy operation and a wide range of functions.Adenoviruses are one of the most common used vectors,inducing strong immune as well as inflammatory reactions.Oncolytic virotherapy,using the above mentioned in vivo viral vectors,is one of the most promising nonpathogenic,highly-selective cytotoxic anti-cancer therapy using anti-cancer agents with high anti-tumor potency and strong oncolytic effect.There have been a variety of targeted therapeutic and pre-clinical strategies tested for gene therapy in pancreatic cancer such as gene-editing systems(e.g.,clustered regularly interspaced palindromic repeats-Cas9),RNA interference technology(e.g.,microRNAs,short hairpin RNA or small interfering RNA),adoptive immunotherapy and vaccination(e.g.,chimeric antigen receptor T-cell therapy)with encouraging results.

Cytochrome P450 Directed Prodrug Activation Therapy in Research of Cancer Enzymology

Cancer enzymology is a promising filiation of bio-medical sciences. In thepast decades, enzymes, such as GST(glutathione S-transferase) , PKC(protein kinase C) , Topo(DNAtopoisomerases), TK(tyrosine kinase), CD (bacterial cytosine deaminase), CPG2(carboxypeptidase G2) ,and PNP (purine nucleoside phosphorylase), have been known to bear close relations to cancer. Theirspecific expression and influence on the process of tumor initiation, promotion and progressionattract scientists to apply them as a biochemical marker of certain malignant tumor, a predictor ofresponse in cancer chemotherapy; to apply them to drug design, tumor prevention and as adjuvant toradiotherapy or surgery.

Micelles as potential drug delivery systems for colorectal cancer treatment

Despite the significant progress in cancer therapy,colorectal cancer(CRC)remains one of the most fatal malignancies worldwide.Chemotherapy is currently the mainstay therapeutic modality adopted for CRC treatment.However,the long-term effectiveness of chemotherapeutic drugs has been hampered by their low bioavailability,non-selective tumor targeting mechanisms,non-specific biodistribution associated with low drug concentrations at the tumor site and undesirable side effects.Over the last decade,there has been increasing interest in using nanotechnology-based drug delivery systems to circumvent these limitations.Various nanoparticles have been developed for delivering chemotherapeutic drugs among which polymeric micelles are attractive candidates.Polymeric micelles are biocompatible nanocarriers that can bypass the biological barriers and preferentially accumulate in tumors via the enhanced permeability and retention effect.They can be easily engineered with stimuli-responsive and tumor targeting moieties to further ensure their selective uptake by cancer cells and controlled drug release at the desirable tumor site.They have been shown to effectively improve the pharmacokinetic properties of chemotherapeutic drugs and enhance their safety profile and anticancer efficacy in different types of cancer.Given that combination therapy is the new strategy implemented in cancer therapy,polymeric micelles are suitable for multidrug delivery and allow drugs to act concurrently at the action site to achieve synergistic therapeutic outcomes.They also allow the delivery of anticancer genetic material along with chemotherapy drugs offering a novel approach for CRC therapy.Here,we highlight the properties of polymeric micelles that make them promising drug delivery systems for CRC treatment.We also review their application in CRC chemotherapy and gene therapy as well as in combination cancer chemotherapy.

Insight to drug delivery aspects for colorectal cancer

Colorectal cancer(CRC) is the third most common cancer diagnosed worldwide in human beings. Surgery, chemotherapy, radiotherapy and targeted therapiesare the conventional four approaches which are currently used for the treatment of CRC. The site specific delivery of chemotherapeutics to their site of action would increase effectiveness with reducing side effects. Targeted oral drug delivery systems based on polysaccharides are being investigated to target and deliver chemotherapeutic and chemopreventive agents directly to colon and rectum. Site-specific drug delivery to colon increases its concentration at the target site, and thus requires a lower dose and hence abridged side effects. Some novel therapies are also briefly discussed in article such as receptor(epidermal growth factor receptor, folate receptor, wheat germ agglutinin, VEGF receptor, hyaluronic acid receptor) based targeting therapy; colon targeted proapoptotic anticancer drug delivery system, gene therapy. Even though good treatment options are available for CRC, the ultimate therapeutic approach is to avert the incidence of CRC. It was also found that CRCs could be prevented by diet and nutrition such as calcium, vitamin D, curcumin, quercetin and fish oil supplements. Immunotherapy and vaccination are used nowadays which are showing better results against CRC.

Design of Dendrimer Modified Carbon Nanotubes for Gene Delivery

To investigate the efficiency of polyamidoamine dendrimer grafted carbon nanotube （dendrimer-CNT） mediated entrance of anti-survivin oligonucleotide into MCF-7 cells, and its effects on the growth of MCF-7 cells. Methods： Antisense survivin oligonucleotide was anchored onto polyamidoamine dendrimer grafted carbon nanotubes to form dendrimer-CNT-asODN complex and the complex was characterized by Zeta potential, AFM, TEM, and 1% agarose gel electrophoresis analysis. Dendrimer-CNT-asODN complexes were added into the medium and incubated with MCF-7 cells. MTT method was used to detect the effects of asODN and dendrimer-CNT-asODN on the growth of MCF-7 cells. TEM was used to observe the distribution of dendrimer-CNT-asODN complex within MCF-7 cells. Results： Successful synthesis of dendrimer-CNT-asODN complexes was proved by TEM, AFM and agarose gel electrophoresis. TEM showed that the complexes were located in the cytoplasm, endosome, and lysosome within MCF-7 cells. When dendrimer-CNT-asODN （1.0 μmol/L） and asODN （1.0 μmol/L） were used for 120 h incubation, the inhibitory rates of MCF-7 cells were （28.22±3.5）% for dendrimer-CNT-asODN complex group, （9.23±0.56）% for only asODN group, and （3.44±0.25）% for dendrimer-CNT group. Dendrimer-CNT-asODN complex at 3.0 μmol/L inhibited MCF-7 cells by （30.30±10.62）%, and the inhibitory effects were in a time- and concentration-dependent manner. Conclusion： Dendrimer-CNT nanoparticles may serve as a gene delivery vector with high efficiency, which can bring foreign gene into cancer cells, inhibiting cancer cell proliferation and markedly enhancing the cancer therapy effects.

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.

M1-polarized macrophage-derived cellular nanovesicle-coated lipid nanoparticles for enhanced cancer treatment through hybridization of gene therapy and cancer immunotherapy

Optimum genetic delivery for modulating target genes to diseased tissue is a major obstacle for profitable gene therapy.Lipid nanoparticles(LNPs),considered a prospective vehicle for nucleic acid delivery,have demonstrated efficacy in human use during the COVID-19 pandemic.This study introduces a novel biomaterial-based platform,M1-polarized macrophage-derived cellular nanovesicle-coated LNPs(M1-C-LNPs),specifically engineered for a combined gene-immunotherapy approach against solid tumor.The dual-function system of M1-C-LNPs encapsulates Bcl2-targeting siRNA within LNPs and immune-modulating cytokines within M1 macrophage-derived cellular nanovesicles(M1-NVs),effectively facilitating apoptosis in cancer cells without impacting T and NK cells,which activate the intratumoral immune response to promote granule-mediating killing for solid tumor eradication.Enhanced retention within tumor was observed upon intratumoral administration of M1-C-LNPs,owing to the presence of adhesion molecules on M1-NVs,thereby contributing to superior tumor growth inhibition.These findings represent a promising strategy for the development of targeted and effective nanoparticle-based cancer genetic-immunotherapy,with significant implications for advancing biomaterial use in cancer therapeutics.

Engineering nanotheranostic strategies for liver cancer

The incidence and mortality of hepatocellular carcinoma have continued to increase over the last few years,and the medicine-based outlook of patients is poor.Given great ideas from the development of nanotechnology in medicine,especially the advantages in the treatments of liver cancer.Some engineering nanoparticles with active targeting,ligand modification,and passive targeting capacity achieve efficient drug delivery to tumor cells.In addition,the behavior of drug release is also applied to the drug loading nanosystem based on the tumor microenvironment.Considering clinical use of local treatment of liver cancer,in situ drug delivery of nanogels is also fully studied in orthotopic chemotherapy,radiotherapy,and ablation therapy.Furthermore,novel therapies including gene therapy,phototherapy,and immunotherapy are also applied as combined therapy for liver cancer.Engineering nonviral polymers to function as gene delivery vectors with increased efficiency and specificity,and strategies of co-delivery of therapeutic genes and drugs show great therapeutic effect against liver tumors,including drug-resistant tumors.Phototherapy is also applied in surgical procedures,chemotherapy,and immunotherapy.Combination strategies significantly enhance therapeutic effects and decrease side effects.Overall,the application of nanotechnology could bring a revolutionary change to the current treatment of liver cancer.

RNA interference against hypoxia inducible factor-1α inhibited growth of cervical cancer by limiting vascularization

Objective:Cervical cancer has become a major public health problem.The development of effective,systemic therapies for cervical cancer is highly desired.We show here that hypoxia inducible factor-1α(HIF-1α) was indicated as an attractive therapeutic molecular target for cervical cancer.Methods:Firstly,we observed the expressional level of HIF-1α in cervical cancer and Hela and Siha cell lines.Secondly,by constructuring HIF-1α shRNA targeting human HIF-1α mRNA common sequence and transfecting it with plasmid to cervical cell,we detected the changes of HIF-1α and its downstream genes levels VEGF.Then we injected selected stably transfected cell line into athymic nude mice to estimate its' antitumor effects.Results:We observed that HIF-1α inhibition was related to down-regulated VEGF resulting in prevention of angiogenesis,then leading to slower-growing tumors.Conclusion:The underlying concept of transfecting a HIF-1α shRNA expression vector to block the HIF-1α holds promise as the clinical potential of gene therapy for cervical cancer.

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.

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.

Folate-conjugated polyspermine for lung cancer–targeted gene therapy

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors.Spermine(SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol)(PEG)diacrylate(SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid(FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector(FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamines2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors(FRs). Moreover, in vivo delivery of green fluorescent protein(GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.

Pseudotyped lentiviral vectors:Ready for translation into targeted cancer gene therapy?

Gene therapy holds great promise for curing cancer by editing the deleterious genes of tumor cells,but the lack of vector systems for efficient delivery of genetic material into specific tumor sites in vivo has limited its full therapeutic potential in cancer gene therapy.Over the past two decades,increasing studies have shown that lentiviral vectors(LVs)modified with different glycoproteins from a donating virus,a process referred to as pseudotyping,have altered tropism and display cell-type specificity in transduction,leading to selective tumor cell killing.This feature of LVs together with their ability to enable high efficient gene delivery in dividing and non-dividing mammalian cells in vivo make them to be attractive tools in future cancer gene therapy.This review is intended to summarize the status quo of some typical pseudotypings of LVs and their applications in basic anti-cancer studies across many malignancies.The opportunities of translating pseudotyped LVs into clinic use in cancer therapy have also been discussed.

Enhanced endosomal escape of dendrigraft poly-L-lysine polymers for the efficient gene therapy of breast cancer

Dendrimer,such as dendrigraft poly-L-lysine(DGL)polymers,with high surface charge density,well-defined structure,and narrow poly-dispersity is often employed as a gene vector,but its transfection efficiency is still partially inhibited due to poor endosomal escape ability.Herein,we used a surface modification strategy to enhance the endosomal escape ability of DGL polymers,and thus improved its gene transfection efficiency.A library of phenylboronic acid(PBA)modified DGL polymers(PBA-DGLs)was designed to screen efficient small interfering RNA(siRNA)vectors.The lead candidate screened from the library shown a capability of inducing nearly 90% gene silencing in MDA-MB-231 cells.The study of the transfection mechanism revealed that PBA modification not only improves siRNA cellular uptake,but,more importantly,endows DGL polymers the ability of endosomal escape.One of the top candidates from polyplexes was further shielded with hyaluronic acid to construct targeted nanoparticles,and the yielding nanoparticles significantly suppressed the tumor growth in a breast cancer model by effective siRNA delivery.This research provides a general and effective strategy to enhance the endosomal escape and transfection efficiency of dendrimer.