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.

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 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.

Macrophage-mediated nanoparticle delivery to the periodontal lesions in established murine model via Pg-LPS induction.

We established a murine periodontitis model by local injection of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) into the gingival sulcus of mandibular left incisor four times with 48-h interval. The histological examination of the periodontal tissues demonstrated that significant loss of periodontal bone and ligaments was observed in the lesion side with abundant inflammatory cell infiltration. Two days after the last injection, Cy5-labelled siRNA/chitosan particles were injected intraperitoneally (ip). The chitosan/siRNA particles were taken up by peritoneal macrophages, which subsequently migrated to the inflamed gingival area evaluated by in vivo imaging. The localization of macrophages in the inflamed region was further confirmed by immunofluorescent staining. The present report demonstrates that intragingival injection of Pg-LPS can be used to create an experimental model of periodontal inflammation in mice and that recruitment of macrophages with chitosan/siRNA nanoparticles to the inflamed area opens the possibility of an RNAi-based therapeutic approach using chitosan as a carrier in periodontitis.

Sialic acid residues are essential for cell lysis mediated by leukotoxin from Aggregatibacter actinomycetemcomitans.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

Chitosan hydrogel as siRNA vector for prolonged gene silencing.

BACKGROUND: The periodontitis is one of the most prevalent diseases with alveolar resorption in adult people and is the main cause of the tooth loss. To investigate the possibility for protecting the loss of alveolar bone in periodontal diseases, a RNAi-based therapeutic strategy is applied for silencing RANK signaling using thermosensitive chitosan hydrogel as siRNA reservoir and vector. RESULTS: The thermosensitive chitosan hydrogel was formed from solution (PH = 7.2, at 4°C) at 37°C within 8 minutes. The degradation rates of hydrogel were ~50% and 5% (W remaining/W beginning) in the presence and absence of lysozyme, respectively, over a period of 20 days. The concurrent cumulative in vitro release of Cy3-labeled siRNA from the hydrogel was 50% and 17% over 14 days, with or without lysozyme digestion, respectively. High cell viability (>88%) was maintained for cells treated with hydrogel loaded with RANK specific siRNA and RANK knockdown was prolonged for up to 9 days when cells were incubated with siRNA/hydrogel complex. In vivo release of siRNA was investigated in a subcutaneous delivery setup in mice. The fluorescent signal from siRNA within hydrogel was remained for up to 14 days compared to less than one day for siRNA alone. CONCLUSIONS: Chitosan hydrogel can potentially serve as a suitable reservoir and vector for local sustained delivery of siRNA in potential therapy.

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.

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.

Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to the lesion site in a murine radiation-induced fibrosis model.

BACKGROUND: Radiation-induced fibrosis (RIF) is a dose-limiting complication of cancer radiotherapy and causes serious problems, i.e. restricted tissue flexibility, pain, ulceration or necrosis. Recently, we have successfully treated RIF in a mouse model by intraperitoneal administration of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. METHODS: Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. RESULTS: We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. CONCLUSION: We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan/siRNA nanoparticle may constitute a general treatment for inflammatory diseases using the natural homing potential of macrophages to inflammatory sites.

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.

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.

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.

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.

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.

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.

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 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.

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.