Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics.

Development of ionizable lipid nanoparticles and a lyophilized formulation for potent CRISPR-Cas9 delivery and genome editing.

CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.

Chitosan functionalized gold nanostars as a theranostic platform for intracellular microRNA detection and photothermal therapy.

The development of a theranostic platform that integrates both diagnostic and therapeutic capabilities is in great need for precise and personalized medicine. Here, we present a novel nanoplatform (AuNS@CS-hpDNA) formulated by chitosan functionalized gold nanostar composites and further complexed with fluorescent hairpin DNA (hpDNA) probes for tumor-related miRNA imaging and photothermal therapy (PTT). The optimized AuNS@CS-hpDNA nanoplatform mediated efficient hpDNA probe loading and intracellular delivery. Subsequently, the cytosol transfer of the hpDNA probe enabled specific hybridization using the targeted miRNA, which triggered the recovery of fluorescence for the precise detection of biomarker miR21 in living cells and realized the distinguishing cancer cell line MCF-7 and normal cells. Meanwhile, the AuNS@CS-hpDNA nanoplatform exhibited excellent photothermal conversion properties, which induced efficient cancer cell killing under laser irradiation. Thus, the developed AuNS@CS-hpDNA nanoplatform could simultaneously realize the precise detection of cancer cells and accurately initiate efficient PTT, which represents a promising strategy for precise cancer therapy.

Engineered ionizable lipid nanoparticles mediated efficient siRNA delivery to macrophages for anti-inflammatory treatment of acute liver injury.

Small interfering RNA (siRNA) mediating specific gene silencing provides a promising strategy for anti-inflammatory therapy. However, the development of potent carriers for anti-inflammatory siRNA to macrophages remains challenging. With the aim of realizing potent delivery of siRNA to macrophages, we engineered ionizable lipid nanoparticles (LNPs) with the key component of synthetic lipid-like materials. By varying the amine molecules in the structure of synthetic lipid-like materials, a potent LNP (1O14-LNP) was identified, which exhibited efficient transfection of macrophages by facilitating efficient internalization and endosomal escape. The 1O14-LNP successfully delivered anti-inflammatory siRNA against interleukin-1ß (siIL-1ß) with more than 90% downregulation of IL-1ß expression in LPS-activated macrophages. From in vivo studies, systemic administrated 1O14-LNP/siRNA mainly distributed in liver and efficiently captured by hepatic macrophages without notable sign of toxicity. Furthermore, LPS/d-GalN-induced acute liver injury model treated with 1O14-LNP/siIL-1ß resulted in significant suppression of IL-1ß expression and amelioration of liver tissue damage. These results demonstrate that the engineered ionizable LNP provides a powerful tool for siRNA delivery to macrophages and that the strategy of silencing of pro-inflammatory cytokines holds great potential for treating inflammatory diseases.

Niosome-Assisted Delivery of DNA Fluorescent Probe with Optimized Strand Displacement for Intracellular MicroRNA21 Imaging.

MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system to achieve fluorescence imaging of miRNA21 (miR21) in cancer cells. This system consists of two components: an optimized "off-on" double-stranded DNA (dsDNA) fluorescent for miR21 sensing by efficient strand-displacement reaction and a potent carrier vesicle, termed niosome (SPN), to facilitate the efficient intracellular delivery of the dsDNA probe. A series of dsDNA probes based on fluorescence energy resonance transfer (FRET) was assembled to target miR21. By optimizing the appropriate length of the reporter strand in the dsDNA probe, high accuracy and sensitivity for miR21 recognition are ensured. To overcome the cellular barrier, we synthesized SPN with the main components of a nonionic surfactant Span 80 and a cationic lipid DOTAP, which could efficiently load dsDNA probes via electrostatic interactions and potently deliver the dsDNA probes into cells with good biosafety. The SPN/dsDNA achieved efficient miR21 fluorescent imaging in living cells, and could discriminate cancer cells (MCF-7) from normal cells (L-02). Therefore, the proposed SPN/dsDNA system provides a powerful tool for intracellular miRNA biosensing, which holds great promise for early cancer diagnosis.

Boosting ionizable lipid nanoparticle-mediated in vivo mRNA delivery through optimization of lipid amine-head groups.

In vitro transcribed messenger RNA (IVT-mRNA) holds great promise for the development of novel therapeutics, such as immunotherapy and vaccination. However, the main obstacle towards clinical translation is the lack of effective delivery systems. Herein, we have synthesized a series of ionizable lipids by the addition of an alkyl-acrylate to amine-containing molecules (amine-head groups) as a key component of ionizable lipid nanoparticles (iLNPs) and thoroughly investigated the impact of the amine-head group on the transfection efficiency of iLNPs/mRNA lipoplexes both in vitro and in vivo. The top-performing iLNP (114-iLNP), composed of a lipid with spermine as the amine-head, demonstrated the strongest cellular uptake, membrane disruption and endosomal escape, and further achieved the highest protein expression in HeLa cells with more than 95% transfection efficiency. More importantly, intravenous injection of luciferase mRNA loaded 114-iLNP enables the most efficacious in vivo protein expression, predominantly in the liver. Biodistribution and biosafety evaluation of 114-iLNP/mRNA further demonstrated the liver-selective delivery capability and high biocompatibility. In addition, 114-iLNP facilitated efficient in vivo delivery of a therapeutic gene, human erythropoietin (hEPO) mRNA, and induced hEPO expression in a dose-dependent manner. Therefore, these results demonstrate that the amine-head group in the ionizable lipid significantly affects mRNA delivery efficacy and the leading candidate 114-iLNP composed of a lipid with spermine as the amine-head has great potential for mRNA therapeutics development.

Identification of a potent ionizable lipid for efficient macrophage transfection and systemic anti-interleukin-1ß siRNA delivery against acute liver failure.

RNA interference (RNAi) therapy has great potential for treating inflammatory diseases. However, the development of potent carrier materials for delivering siRNA to macrophages is challenging. Herein, we design a set of ionizable lipid nanoparticles (LNPs) to screen and identify a potent carrier of siRNA for silencing an essential pro-inflammatory cytokine, interleukin-1ß (IL-1ß) in macrophages. The top performance LNP (114-LNP), containing ionizable lipid with spermine as an amine-head group, facilitated efficient siRNA internalization via multiple endocytosis pathways and achieved effective endosome escape in macrophages. The optimized LNP/siIL-1ß achieved strong silencing of IL-1ß in both activated Raw 264.7 cells and primary macrophages. Furthermore, systematic administration of 114-LNP/siIL-1ß complexes could effectively inhibit IL-1ß expression in an acute liver failure model and significantly attenuated hepatic inflammation and liver damage. These results suggest that the optimized ionizable lipid nanoparticle represents a promising platform for anti-inflammation therapies.

Identification and Nanomechanical Characterization of the HIV Tat-Amyloid ß Peptide Multifibrillar Structures.

HIV transactivator of transcription (Tat) protein could interact with amyloid ß (Aß) peptide which cause the growth of Aß plaques in the brain and result in Alzheimer's disease in HIV-infected patients. Herein, we employ high-resolution atomic force microscopy and quantitative nanomechanical mapping to investigate the effects of Tat protein in Aß peptide aggregation. Our results demonstrate that the Tat protein could bind to the Aß fibril surfaces and result in the formation of Tat-Aß multifibrillar structures. The resultant Tat-Aß multifibrillar aggregates represent an increase in stiffness compared with Aß fibrils due to the increase in ß-sheet formation. The identification and characterization of the Tat-Aß intermediate aggregates is important to understanding the interactions between Tat protein and Aß peptide, and the development of novel therapeutic strategy for Alzheimer's disease-like disorder in HIV infected individuals.

Acoustically responsive polydopamine nanodroplets: A novel theranostic agent.

Ultrasound-induced cavitation has been used as a tool of enhancing extravasation and tissue penetration of anticancer agents in tumours. Initiating cavitation in tissue however, requires high acoustic intensities that are neither safe nor easy to achieve with current clinical systems. The use of cavitation nuclei can however lower the acoustic intensities required to initiate cavitation and the resulting bio-effects in situ. Microbubbles, solid gas-trapping nanoparticles, and phase shift nanodroplets are some examples in a growing list of proposed cavitation nuclei. Besides the ability to lower the cavitation threshold, stability, long circulation times, biocompatibility and biodegradability, are some of the desirable characteristics that a clinically applicable cavitation agent should possess. In this study, we present a novel formulation of ultrasound-triggered phase transition sub-micrometer sized nanodroplets (~400 nm) stabilised with a biocompatible polymer, polydopamine (PDA). PDA offers some important benefits: (1) facile fabrication, as dopamine monomers are directly polymerised on the nanodroplets, (2) high polymer biocompatibility, and (3) ease of functionalisation with other molecules such as drugs or targeting species. We demonstrate that the acoustic intensities required to initiate inertial cavitation can all be achieved with existing clinical ultrasound systems. Cell viability and haemolysis studies show that nanodroplets are biocompatible. Our results demonstrate the great potential of PDA nanodroplets as an acoustically active nanodevice, which is highly valuable for biomedical applications including drug delivery and treatment monitoring.

Lipidoid-siRNA Nanoparticle-Mediated IL-1ß Gene Silencing for Systemic Arthritis Therapy in a Mouse Model.

Interleukin-1 beta (IL-1ß) plays a central role in the induction of rheumatoid arthritis (RA). In the present study, we demonstrated that lipidoid-polymer hybrid nanoparticle (FS14-NP) can efficiently deliver siRNA against IL-1ß (siIL-1ß) to macrophages and effectively suppress the pathogenesis of experimental arthritis induced by collagen antibody (CAIA mice). FS14-NP/siIL-1ß achieved approximately 70% and 90% gene-silencing efficiency in the RAW 264.7 cell line and intraperitoneal macrophages, respectively. Intravenous administration of FS14-NP/siRNA led to rapid accumulation of siRNA in macrophages within the arthritic joints. Furthermore, FS14-NP/siIL-1ß treatment lowered the expression of pro-inflammatory cytokines in arthritic joints and dramatically attenuated ankle swelling, bone erosion, and cartilage destruction. These results demonstrate that FS14-NP/siIL-1ß may represent an effective therapy for systemic arthritis and other inflammatory disorders.

Selective Delivery of Doxorubicin to EGFR+ Cancer Cells by Cetuximab-DNA Conjugates.

Doxorubicin is a hydrophobic anticancer drug that has poor selectivity, due to the lack of active targeting capability. Here, learning lessons from the success of antibody-drug conjugates, we have designed a new doxorubicin delivery system without conjugating doxorubicin to antibody directly. In this setup, cetuximab, an antibody that targets the epidermal growth factor receptor (EGFR) in cancer cells, was conjugated to a single-stranded DNA with a carefully designed sequence in a site-selective manner by using the DNA-templated protein conjugation (DTPC) method. The DNA duplex in the conjugates serves as a carrier of doxorubicin through noncovalent intercalation, and cetuximab functions as the targeting agent; this could drastically decrease systemic toxicity and potentially avoid under- or overdosing. The size of conjugates loaded with doxorubicin was about 8.77 or 16.61 nm when characterized by dynamic light scattering and atomic force microscopy, respectively. In vitro cytotoxicity and selective cancer cell killing was investigated against two EGFR+ cell lines (KB and MDA-MB-231) and one EGFR- cell line (NIH-3T3). Cytotoxicity and flow cytometry data showed that doxorubicin loaded in cetuximab-DNA conjugates was more potent in terms of cell cytotoxicity than free doxorubicin in EGFR-overexpressed cell lines, thus suggesting that the conjugates were more selectively and easily taken up into cells, followed by rapid release of doxorubicin from the system into the cytoplasm from endosomes.

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG-containing niosome system (iSPN) demonstrated highly efficient siRNA and miRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient near-infrared labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.

Calcium-MicroRNA Complex-Functionalized Nanotubular Implant Surface for Highly Efficient Transfection and Enhanced Osteogenesis of Mesenchymal Stem Cells.

Controlling mesenchymal stem cell (MSC) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible, and highly effective delivery method of small RNA therapeutics into human MSCs (hMSCs) from an implant surface by calcium ions. First, we demonstrated that simple Ca/siRNA targeting green fluorescent protein (GFP) nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca2+ concentration (>5 mM). In addition, a single transfection could obtain a long-lasting silencing effect for more than 2 weeks. All three of the main endocytosis pathways (clathrin- and caveolin-mediated endocytosis and macropinocytosis) were involved in the internalization of the Ca/siRNA complexes by MSCs, and macropinocytosis plays the most dominant role. Furthermore, the Ca/siRNA complexes could be efficiently loaded onto the titanium implant surface when pretreated with anodization to create a nanotube (NT) layer. Because of the hydrophilic property of the NT surface, the Ca/siRNA was quickly loaded (less than 4 h) with high efficiency (nearly 100%), forming an even amorphous coating. The Ca/siRNA-coated NT surface showed an initial burst release of 80% of the siRNA complexes over 2 h, which is adequate to achieve robust gene silencing of attached hMSCs. To demonstrate the therapeutic potential of our Ca/siRNA coating technology, Ca/antimiR-138 complexes were loaded on to the NT surface, which strongly enhanced the osteogenic differentiation of hMSCs. In conclusion, our findings suggest that Ca2+ is an effective and biocompatible carrier to deliver small RNA therapeutics into hMSCs, both in solution and from functionalized surfaces, which provides a novel approach to control the MSC differentiation and tissue regeneration.

Theranostic poly(lactic-co-glycolic acid) nanoparticle for magnetic resonance/infrared fluorescence bimodal imaging and efficient siRNA delivery to macrophages and its evaluation in a kidney injury model.

In this work, a theranostic nanoparticle was developed for multimodal imaging and siRNA delivery. The core of the nanoparticles (NP) was formed by encapsulation of superparamagnetic iron oxides and indocyanine green in a PLGA matrix to serve as a multimodal probe for near-infrared (NIFR) and magnetic resonance (MR) imaging. The surface of the particle was coated with polyethylenimine (PEI) for siRNA delivery. Macrophages efficiently took up the nanoparticles and emitted strong NIFR and MR contrast. When transfected with siRNA targeting the pro-inflammatory enzyme cyclooxygenase-2 (COX-2), significant down-regulation of COX-2 was achieved in activated macrophages. Furthermore, after injection into a unilateral ureteral obstruction (UUO)-induced kidney injury model, NIFR and MRI imaging revealed accumulation of nanoparticles in the injury kidney. In addition, in vivo silencing of COX-2 was achieved by NP/PEI/siCOX-2, which further attenuated kidney injury. Our theranostic platform represents a promising approach for simultaneous diagnosis and treatment of inflammatory diseases.

Autophagy plays a dual role during intracellular siRNA delivery by lipoplex and polyplex nanoparticles.

Growing evidence indicates that autophagy plays a vital role during intracellular DNA delivery mediated by lipoplex and polyplex nanoparticles. However, autophagy in intracellular siRNA delivery has not been well understood. In this study, lipofectamine 2000 and chitosan were used to formulate lipoplex and polyplex with siRNA for systematically investigating the interplay between siRNA delivery and autophagy. After transfection of H1299 cells with lipoplex and polyplex, the number of autophagic vacuoles was increased significantly indicated by the accumulation of monodansylcadaverine (MDC) staining. Western blot revealed that the LC3-II expression was significantly increased after transfection, whereas p-mTOR expression was not influenced apparently. In addition, small-molecule autophagy modulators significantly affected transfection efficiency. Specifically, the mTOR-dependent autophagy inducer rapamycin enhanced the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine (3-MA) suppressed their silencing efficiency. On the contrary, mTOR-independent autophagy inducer LiBr decreased whereas mTOR-independent autophagy inhibitor thapsigargin (TG) increased the knockdown efficacy. Immunofluorescence staining showed that siRNA was partially co-localized with autophagosomes and the percentage of co-localized siRNA was significantly affected by autophagy modulators in the opposite trend of gene knockdown efficacy. In conclusion, our study suggests that autophagy plays an important role during the intracellular siRNA trafficking mediated by both lipoplex and polyplex. Modulating autophagy process will result in distinct knockdown efficiency, which may be applied as a potential convenient way for improving siRNA delivery efficacy. STATEMENT OF SIGNIFICANCE: Although tremendous effects has been made in the development of non-viral siRNA delivery systems, the intracellular siRNA trafficking has not been elucidated clearly. In this study, we systematically investigated the relationship between autophagy and intracellular siRNA delivery. We found that the non-viral siRNA delivery by both lipoplex and polyplex could induce mTOR-independent autophagy response. More interestingly, knockdown efficiency of both lipoplex and polyplex could be modulated with different autophagy regulators. Specifically, the mTOR-dependent autophagy inducer rapamycin enhances the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine suppresses their silencing efficiency. On the contrary, mTOR-independent autophagy inducer lithium bromide decreases, whereas mTOR-independent autophagy inhibitor thapsigargin increases the knockdown efficacy. These findings suggest that the mTOR-dependent and -independent autophagy play a distinct role in the intracellular siRNA trafficking. Furthermore, co-administration with proper autophagy regulators could be potential convenient method to modulate siRNA transfection efficacy.

Impact of PEG Chain Length on the Physical Properties and Bioactivity of PEGylated Chitosan/siRNA Nanoparticles in Vitro and in Vivo.

PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated polyplexes showed distinct mechanism of endocytosis, which was macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. Efficient silence of ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs). Significant extension of in vivo circulation was achieved only with high molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CS-PEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo.

Enhanced efficacy of chemotherapy for breast cancer stem cells by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense.

While chemotherapy is universally recognized as a frontline treatment strategy for breast cancer, it is not always successful; among the leading causes of treatment failure is existing and/or acquired multidrug resistance. Cancer stem cells (CSCs), which constitute a minority of the cells of a tumor, are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ATP-binding cassette transporters (ABC transporters), an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, more effective therapy, especially targeted to CSCs, is urgently required. We studied the characteristics of 231-CSCs (CD44+/CD24-) sorted from human MDA-MB-231 breast cancer cells and demonstrated that 231-CSCs exhibited enhanced capacities for proliferation, migration, tumorigenesis and chemotherapy resistance. To address these multifunctional facets of CSCs, we devised a non-ionic surfactant-based vesicle (niosome) co-delivery system to simultaneously deliver siRNAs, targeted to both the ABC transporter (ABCG2) and the anti-apoptosis defense gene (BCL2), and doxorubicin (DOX) to CSCs. The rationale is to sensitize CSCs to DOX by down regulating the drug-resistance gene ABCG2 and simultaneously induce apoptosis by lowering BCL2 expression. The co-delivery system (CDS) successfully delivered siRNAs and DOX to the cytoplasm and nuclei, respectively, and resulted in a down-regulation of ABCG2- and BCL2 mRNAs in CSCs by 60% and 65%, respectively, compared to the control. A corresponding decrease in protein expression was observed using Western blotting. The IC50 of DOX in CSCs concurrently decreased significantly. Our result established CDS as a promising multi-drug delivery platform for cancer treatment. STATEMENT OF SIGNIFICANCE: Cancer stem cells (CSCs) are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ABC transporters, an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, effective therapy, especially to CSCs, is urgently required. In current study, we studied the characteristics of 231-CSCs sorted from human MDA-MB-231 breast cancer cells and found that 231-CSCs possessed enhanced proliferation, migration, tumorigenesis, and DOX resistance. We employed a non-ionic surfactant-based vesicle (niosome) delivery system to simultaneously deliver siRNAs targeted to multi-drug resistance genes, and DOX to kill 231-CSCs. The CDS showed an enhanced therapeutic effect by resensitizing 231-CSCs to DOX and may constitute a promising candidate for cancer chemotherapy.

Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion.

Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium surfaces with specific gene-regulatory siRNAs to enhance biofunction.

Chitosan/siRNA nanoparticles targeting cyclooxygenase type 2 attenuate unilateral ureteral obstruction-induced kidney injury in mice.

Cyclooxygenase type 2 (COX-2) plays a predominant role in the progression of kidney injury in obstructive nephropathy. The aim of this study was to test the efficacy of chitosan/small interfering RNA (siRNA) nanoparticles to knockdown COX-2 specifically in macrophages to prevent kidney injury induced by unilateral ureteral obstruction (UUO). Using optical imaging techniques and confocal microscopy, we demonstrated that chitosan/siRNA nanoparticles accumulated in macrophages in the obstructed kidney. Consistent with the imaging data, the obstructed kidney contained a higher amount of siRNA and macrophages. Chitosan-formulated siRNA against COX-2 was evaluated on RAW macrophages demonstrating reduced COX-2 expression and activity after LPS stimulation. Injection of COX-2 chitosan/siRNA nanoparticles in mice subjected to three-day UUO diminished the UUO-induced COX-2 expression. Likewise, macrophages in the obstructed kidney had reduced COX-2 immunoreactivity, and histological examination showed lesser tubular damage in COX-2 siRNA-treated UUO mice. Parenchymal inflammation, assessed by tumor necrosis factor-alpha (TNF-α) and interleukin 6 mRNA expression, was attenuated by COX-2 siRNA. Furthermore, treatment with COX-2 siRNA reduced heme oxygenase-1 and cleaved caspase-3 in UUO mice, indicating lesser oxidative stress and apoptosis. Our results demonstrate a novel strategy to prevent UUO-induced kidney damage by using chitosan/siRNA nanoparticles to knockdown COX-2 specifically in macrophages.

Ultrastable green fluorescence carbon dots with a high quantum yield for bioimaging and use as theranostic carriers.

Carbon dots (Cdots) have recently emerged as a novel platform of fluorescent nanomaterials. These carbon nanoparticles have great potential in biomedical applications such as bioimaging as they exhibit excellent photoluminescence properties, chemical inertness and low cytotoxicity in comparison to widely used semiconductor quantum dots. However, it remains a great challenge to prepare highly stable, water-soluble green luminescent Cdots with a high quantum yield. Herein we report a new synthesis route for green luminescent Cdots imbuing these desirable properties and demonstrate their potential in biomedical applications. Oligoethylenimine (OEI)-ß-cyclodextrin (ßCD) Cdots were synthesised using a simple and fast heating method in phosphoric acid. The synthesised Cdots showed strong green fluorescence under UV excitation with a 30% quantum yield and exhibited superior stability over a wide pH range. We further assembled the Cdots into nanocomplexes with hyaluronic acid for potential use as theranostic carriers. After confirming that the Cdot nanocomplexes exhibited negligible cytotoxicity with H1299 lung cancer cells, in vitro bioimaging of the Cdots and nanocomplexes was carried out. Doxorubicin (Dox), an anticancer drug, was also loaded into the nanocomplexes and the cytotoxicity effect of Dox loaded nanocomplexes with H1299 lung cancer cells was evaluated. Thus, this work demonstrates the great potential of the novel OEI-ßCD Cdots in bioimaging and as theranostic carriers.