Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

Tumor targeted siRNA delivery by adenosine receptor-specific curdlan nanoparticles.

Aminated curdlan derivatives are highly effective nucleic acid carriers. Previously, we proved that the ligand-functionalized curdlan derivatives have greatly enhanced cell type specificity induced by receptor-mediated internalization in vitro. In this study, to improve biocompatibility and enhance tumor-targeting efficacy of the curdlan derivative, we pegylated the adenosine functionalized amino curdlan derivative (denoted by pAVC polymer). We confirmed that the uptake of pAVC polymer carrying siRNA by tumor cells was adenosine receptor (AR)-dependent and was specifically inhibited by AMP but not by GMP. The pAVC polymers not only preserved the receptor recognition and exhibited significantly decreased cytotoxicity but also showed remarkable tumor targeting efficiency in vivo. The nanoparticles formulated from siRNA (against STAT3) and pAVC4 polymer, which bears the highest degree of PEG substitution, delivered siRNA highly specifically to tumor tissue, knocked down STAT3, and inhibited tumor growth. The pAVC polymers may be a promising carrier for tumor specific delivery of nucleic acid drugs.

Antibody responses to mRNA versus non-mRNA COVID vaccines among the Mongolian population.

Background: A nationwide vaccination program against coronavirus disease 2019 (COVID-19) was started in Mongolia 4 months after the first local transmission, which occurred in November 2020. Previous studies have reported that two doses of COVID-19 vaccine result in increased antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A study was conducted in Mongolia 2 weeks after the second vaccine dose. In the present study, the serum levels of antibodies of individuals 6 months after natural SARS-CoV-2 infection were compared with those of individuals who had not been infected or had been infected but had received two doses of vaccine, including BNT162b2, ChAdOx1 n-CoV-19, Gam-COVID-Vac, and BBIBP-CorV, which were used for COVID-19 in Mongolia. Methods: Of the 450 participants in this study, 237 (52.66%) were female and 213 (47.33%) were male. Four hundred people with or without SARS-CoV-2 infection who received two doses of 4 different COVID-19 vaccine participated in the vaccine groups and vaccine plus SARS-CoV-2 infection groups (50 in each group) and 50 individuals previously infected with SARS-CoV-2 participated in the unvaccinated group. Total antibody against SARS-CoV-2 infection, anti-SARS-CoV-2 N and S protein human IgG, and antibody inhibiting RBD-ACE2 binding were tested. Results: In the BNT162b2 vaccine group, total antibody against SARS-CoV-2 remained constant until 6 months, while the other vaccine groups showed a significant decrease, as compared to the unvaccinated group. The level of anti-SARS-CoV-2 S-RBD protein IgG was significantly increased in the ChAdOx1 n-CoV-19, Gam-COVID-Vac, and BNT162b2 vaccines groups as compared to the unvaccinated group. Participants in the BNT162b2 vaccine group had higher ACE2 inhibition efficiency compared to the other vaccine groups as well as the unvaccinated group. Conclusions: The BNT162b2 vaccine showed the highest level of antibody against SARS-CoV-2, followed by the BBIBP-CorV, Gam-COVID-Vac, and ChAdOx1 n-CoV-19 vaccines. The level of antibodies was increased in people infected with SARS-CoV-2 after vaccination, as compared to uninfected but vaccinated individuals.

Analysis and identification of key anti-inflammatory molecules in Eerdun Wurile and exploration of their mechanism of action in microglia.

Ethnomedicine Eerdun Wurile (EW) can significantly promote poststroke neuro-recovery through modulation of microglia polarization. Fraction 4-6 (F4-6) isolated from EW via serial fractionation inhibits the expression of pro-inflammatory genes in LPS stimulated microglia. However, the key active molecules of F4-6 have not been identified. Herein, we identified alantolactone (Ala) and dehydrodiisoeugenol (Deh) as the active anti-inflammatory components of F4-6 by UPLC-qTof MS analysis. We confirmed that, F4-6, Ala, Deh and mixture of Ala and Deh (Mix) downregulate the expression of several pro-inflammatory genes including Ccl2, Cox2 and Il6 in LPS-treated microglia in a similar pattern. At the same time upregulate the expression of anti-inflammatory genes including Hmox1, Tgfß, Igf1 and Creb1. Moreover, the conditioned culture media obtained from F4-6 treated microglia significantly enhanced proliferation of N2a cells, and promoted neurite outgrowth possibly through upregulation of Nefh and Dlg4. Mechanistically, F4-6 strongly downregulated the expression of NF-κB p65, while also inhibiting the nuclear translocation of p65, leading to the suppression of transcription of pro-inflammatory genes initiated by NF-κB. Collectively, our data identified and quantified the key chemicals of EW and provide insights into the optimization of the herbal composition for neuroprotection.

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen-glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor.

Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A2B receptor. The internalization of siRNA loaded CuAMP4 by cancer cells was inhibited by free AMP as well as endocytosis inhibitors. Moreover, knockdown of A2BR by siRNA, or pre-treatment of the cells with anti-A2BR antibody, strongly inhibited the cellular uptake of CuAMP4. Our findings confirmed that A2BR can be utilized for cell type specific siRNA delivery, and CuAMP4 NP may be a promising delivery system for cancer cell targeted delivery of therapeutic siRNAs.

Oxidized curdlan activates dendritic cells and enhances antitumor immunity.

Curdlan activates dendritic cells (DCs) and enhances DC-based antitumor immunity. However, hydrophobicity and heterogeneity of curdlan particulates hinder perfect binding of curdlan to dectin-1 receptor, resulting in the reduced activation of antigen presenting cells and limited antitumor effects. Herein, we synthesized partially oxidized curdlan derivative (ß-1,3-polyglucuronic acid, denote PGA). PGA-45 polymer, the reaction product prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO2 systems under acid conditions for 45 min, activated DCs, induced the expression of co-stimulatory molecules and cytokines, and promoted allogenic T cell proliferation as well as the expression of IL-2. Mechanistically, PGA-45 polymer strongly enhanced phosphorylation of IKK-ß and reduced the expression of phosphorylated Akt, suggesting that PGA-45 may activate multiple cell surface receptors such as TLR4 and dectin-1. Administration of tumor lysate pulsed DCs pre-treated with PGA-45 particles induced strong antitumor activity in B16F10 melanoma model. Our data suggest that PGA-45 have strong adjuvant effects for anti-cancer immunity and the design of PGA polymers may provide insights in the development of novel adjuvants for cancer immunotherapy.

Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes.

Photoactive RNA probes have unique advantages in the identification of microRNA (miR) targets due to their ability for efficient conjugation to the target sequences by covalent crosslinking, providing stable miR-mRNA complexes for further analysis. Here, we report a highly efficient and straightforward method for miR target identification that is based on photo-reactive chemical probes and RNA-seq technology (denotes PCP-Seq). UV reactive probes were prepared by incorporating psoralen in the specific position of the seed sequence of miR. Cancer cells that were transfected with the miR probes were treated with UV, following the isolation of poly(A) RNA and sequencing of the transcriptome. Quantitative analysis of RNA-seq reads and subsequent validation by qPCR, dual luciferase assay as well as western blotting confirmed that PCP-Seq could highly efficiently identify multiple targets of different miRs in the lung cancer cell line, such as targets PTTG1 and PTGR1 of miR-29a and ILF2 of miR-34a. Collectively, our data showed that PCP-Seq is a robust strategy for miR targets identification, and unique in the identification of the targets that escape degradation by miRISC and maintain normal cellular level, although their translation is repressed.

Modulation of Microglia Polarization through Silencing of NF-κB p65 by Functionalized Curdlan Nanoparticle-Mediated RNAi.

Microglia polarization plays an important role in poststroke recovery. Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory (M2) polarization of microglia are potential therapeutic strategies for inflammation reduction and neuronal recovery after stroke. Here, we evaluated the central nervous system (CNS)-targeted short interfering RNA (siRNA) delivery ability of functionalized curdlan nanoparticles (CMI) and investigated the nuclear factor-κB (NF-κB) p65 silencing efficiency of CMI-mediated siRNA in microglia, as well as the resulting neuroprotective effect of microglia polarization and neuroprotection in vitro and in vivo. The systemic delivery of NF-κB p65 siRNA (sip65) complexed to CMI nanoparticles in the mouse model of transient middle cerebral artery occlusion (tMCAO) resulted in the distribution of siRNA in microglia and significant silencing in NF-κB p65 in the peri-infarct region. Knockdown of NF-κB p65 resulted in M1 to M2 phenotypic transition of microglia, evidenced by the change in the expression pattern of signature cytokines as well as inducible nitric oxide synthase and CD206. Moreover, the CMI-mediated silencing of p65 increased the density of neurons and decreased pyknosis and edema in the peri-infarct region. Assessment of the neurological deficit score on the Bederson scale revealed a significantly reduced score in the mouse model of tMCAO treated with the sip65/CMI complex. Collectively, our data suggest that CMI nanoparticles are a promising CNS-targeting siRNA delivery system, and NF-κB p65 may be a potential therapeutic target for inflammation reduction and poststroke recovery.

Bioactive components of ethnomedicine Eerdun Wurile regulate the transcription of pro-inflammatory cytokines in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Mongolian medicine Eerdun Wurile (EW) has remarkable neural recovery effect, and has been playing a key role in the clinical treatment of neurological disorders including ischemic stroke in Inner Mongolia Autonomous Region of China. The preliminary pharmacological studies in animal suggested that EW regulates the expression of trophic factors in brain lesion and may also balance the polarization of activated microglia (Gaowa et al., 2018). AIM OF THE STUDY: The pool of leading bioactive chemicals underlying the therapeutic effects of EW has not been identified. Therefore, the mechanism of action of EW is poorly understood. This study was aimed to identify the major group of compounds that contribute to the inhibition of neuroinflammation during stroke recovery through regulation of microglia polarization. MATERIALS AND METHODS: The extracts of EW in different solvents were evaluated for their inhibitory ability of cytokine (IP-10) expression in LPS stimulated BV2 cells. The most effective extract (of petroleum ether extract) was further separated to 18 fractionations on a semi-preparative HPLC column, which were assess for the IP-10 down-regulation efficiency by RT-qPCR. The potent isolate was further fractionated in 12 fractions, which showed fewer peaks. The fraction 6 from this isolates, which remarkably down-regulates cytokines expression including IP-10, TNFα and IL-1ß, was analyzed on UPLC-qTOF MS. The key chemicals were measured for their cytokine inhibition in BV2 cells and mouse primary microglia. RESULTS: After two consecutive fractionating by preparative HPLC, petroleum ether extraction of EW gave 12 fractions with relatively distinctive chromatograms. A particular fraction (fraction 6) preserved the inhibitory effects on expression of pro-inflammatory cytokines including IP-10, TNFα, IL-1ß and iNOS. The result of UPLC-qTOF MS analysis showed that the fraction contains 21 chemicals including costunolide, alantolactone, myristicin and linolenic acid, which significantly down-regulate the expression of key pro-inflammatory cytokines in LPS stimulated BV2 cells as well as mouse primary microglia. CONCLUSION: Collectively our data suggest that the bioactive chemical pool which is responsible for the therapeutic effects of EW can be extracted in petroleum ether, and fractionated to a relatively small multiple components. Such components include known anti-inflammatory chemicals, which may contribute to the possible microglia polarization in brain lesion during the recovery of ischemic stroke.

Alkylation enhances biocompatibility and siRNA delivery efficiency of cationic curdlan nanoparticles.

Cationic curdlan derivatives are a class of promising carriers for nucleic acid delivery including short interfering RNA (siRNA). While our previous studies demonstrated the siRNA delivery efficiency of aminated curdlan derivatives, the associated cytotoxicity issue remained unsolved. To investigate the effects of alkylation on the toxicity as well as the transfection efficiency, we conjugated short alkyl chains to 6-amino-6-deoxy-curdlan (6AC-100). The cytotoxicity of alkylated 6AC-100 derivatives (denote CuVa polymers) decreased with the increase of the degree of substitution (DS). CuVa3, with the highest DS, showed a 50% decreased cytotoxicity compared to 6AC-100 to 6AC-100 at a concentration of 140 µg/mL. The CuVa polymers readily complexed with siRNA to form nanoparticles, and induced significant knockdown of a disease related gene (STAT3) in mouse melanoma cell line B16. However, B16 cells transfected with siSTAT3 complexed to CuVa3 showed the highest phenotypic changes. These findings suggest that CuVa polymers have significantly enhanced biocompatibility and may be a promising delivery system for delivery of therapeutic siRNAs.

Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery.

Developing nucleic acid-based tools to control disease-relevant gene expression in human disorders, such as siRNAs, opens up potential opportunities for therapeutics. Because of their high molecular weight and polyanionic nature, synthetic siRNAs fail to cross biological membranes by passive diffusion and therefore, generally require transmembrane siRNA delivery technologies to access the cytoplasm of target cells. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan derivative 6AC-100 in a regioselective manner to introduce different ratios of imidazole rings in the amino units (denoted as Curimi) and evaluated their siRNA binding ability, cytotoxicity, endosome buffering capacity and siRNA transfection efficiency. The novel curdlan based Curimi polymers formed nanoparticles with siRNA at pH 7.4 in range of 85-105 nm and their size distribution increased along with decreasing pH condition. The zeta potential increased by lowering pH value as well. Curimi polymers showed lower toxicity and higher buffering capacity compared to 6AC-100, and efficiently delivered siRNA against to PLK1 into cancer cells, and subsequently, significantly inhibited target mRNA level. Our result suggested that novel curdlan based Curimi polymers may be used as efficient siRNA carrier for cancer therapy.

Design of Peptidomimetic Functionalized Cholesterol Based Lipid Nanoparticles for Efficient Delivery of Therapeutic Nucleic Acids.

Lipid nanoparticles (LNP) are the most potent carriers for the delivery of nucleic acid-based therapeutics. The first FDA approved a short interfering RNA (siRNA) drug that uses a cationic LNP system for the delivery of siRNA against human transthyretin (hTTR). However, preparation of such LNP involves tedious multi-step synthesis with relatively low yields. In the present study, we synthesized cationic peptidomimetic functionalized cholesterol (denote Chorn) in straightforward chemical approaches with high yield. When formulated with helper lipids, Chorn LNPs complexed with siRNA to form nanoparticles with an average diameter of 150 nm to 200 nm. Chorn LNP mediated transfection of a green fluorescence protein (GFP) expressing plasmid resulted in 60% GFP positive cells. Moreover, Chorn LNP delivered siRNA against polo-like kinase 1 (Plk1), a disease related gene in cancer cells and efficiently suppressed the expression of the gene, resulting in significant morphological changes in the cell nuclei. Our data suggested that cholesterol based cationic LNP, prepared through a robust chemical strategy, may provide a promising siRNA delivery system.

Preparation and cell activities of lactosylated curdlan-triornithine nanoparticles for enhanced DNA/siRNA delivery in hepatoma cells.

A long-anticipated cancer therapy would deliver the right type of therapeutic agents to the target in control with minimal systemic toxicity. The purpose of this study was to prepare lactosylated curdlan-triornithine nanocarriers (CTOLs), and target deliver gene to hepatoma cells. Structures and biophysical properties had been elucidated with physical and chemical methods. The results revealed that those functionalized polymers can completely condense the gene into spherical nanoparticles. Cytotoxicity assay, GFP-pDNA and siRNA transfection in vitro were implemented successively. Observations showed that CTOL 20% with the highest lactose acid substitution degree targeted delivered gene into HepG2 cells over expressing ASGPR receptors and had pretty gene knockdown efficiency over 70%. Meanwhile, the carriers showed excellent biocompatibility. Our studies demonstrated that CTOLs with lower toxicity and higher gene binding capacity may serve as a potential valuable platform that can be tailored to target the liver cancer cells for therapeutic gene.

Receptor-mediated delivery of therapeutic RNA by peptide functionalized curdlan nanoparticles.

Natural carbohydrate polymer-based nanoparticles have great biocompatibility that is required for the safe delivery of various drugs including nucleic acid therapeutics. Herein, we designed curdlan-based nanoparticles for cancer cell targeted delivery of short interfering RNA (siRNA). iRGD peptide conjugated 6-amino-6-deoxy curdlan specifically delivered siRNA to integrin expressing cancer cells. Incubation of cancer cells with free iRGD peptide competitively blocked cellular uptake of the iRGD functionalized curdlan nanoparticles. Chloroquine but not nystatin inhibited cellular uptake of iRGD functionalized curdlan nanoparticles, indicating that the iRGD peptide conjugated curdlan nanoparticles were internalized through the receptor (clathrin)-mediated endocytosis. Moreover, a disease related gene Plk1 was substantially knocked down by siRNA carried by 6AC-iRGD nanoparticles in HepG2 cells. Our data suggested that iRGD functionalized curdlan may provide a biocompatible carrier for siRNA delivery.

Traditional Mongolian medicine Eerdun Wurile improves stroke recovery through regulation of gene expression in rat brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Eerdun Wurile (EW) is one of the key Mongolian medicines for treatment of neurological and cardiological disorders. EW is ranked most regularly used Mongolian medicine in clinic. Components of EW which mainly originate from natural products are well defined and are unique to Mongolian medicine. AIM OF THE STUDY: Although the recipe of EW contains known neuroactive chemicals originated from plants, its mechanism of action has never been elucidated at molecular level. The objective of the present study is to explore the mechanism of neuroregenerative activity of EW by focusing on the regulation of gene expression in the brain of rat model of stroke. MATERIALS AND METHODS: Rat middle cerebral artery occlusion (MCAO) models were treated with EW for 15 days. Then, total RNAs from the cerebral cortex of rat MCAO models treated with either EW or control (saline) were extracted and analyzed by transcriptome sequencing. Differentially expressed genes were analyzed for their functions during the recovery of ischemic stroke. The expression level of significantly differentially expressed genes such as growth factors, microglia markers and secretive enzymes in the lesion was further validated by RT-qPCR and immunohistochemistry. RESULTS: Previously identified neuroactive compounds, such as geniposide (Yu et al., 2009), myristicin (Shin et al., 1988), costunolide (Okugawa et al., 1996), toosendanin (Shi and Chen, 1999) were detected in EW formulation. Bederson scale indicated that the treatment of rat MCAO models with EW showed significantly lowered neurological deficits (p < 0.01). The regional cerebral blood circulation was also remarkably higher in rat MCAO models treated with EW compared to the control group. A total of 186 genes were upregulated in the lesion of rat MCAO models treated with EW compared to control group. Among them, growth factors such as Igf1 (p < 0.05), Igf2 (p < 0.01), Grn (p < 0.01) were significantly upregulated in brain after treatment of rat MCAO models with EW. Meanwhile, greatly enhanced expression of microglia markers, as well as complementary components and secretive proteases were also detected. CONCLUSION: Our data collectively indicated that EW enhances expression of growth factors including Igf1 and Igf2 in neurons and microglia, and may stimulate microglia polarization in the brain. The consequences of such activity include stimulation of neuron growth, hydrolysis and clearance of cell debris at the lesion, as well as the angiogenesis.

Design of Mannose-Functionalized Curdlan Nanoparticles for Macrophage-Targeted siRNA Delivery.

6-Amino-6-deoxy-curdlan is a promising nucleic acid carrier that efficiently delivers plasmid DNA as well as short interfering RNA (siRNA) to various cell lines. The highly reactive C6-NH2 groups of 6-amino-6-deoxy-curdlan prompt conjugation of various side groups including tissue-targeting ligands to enhance cell-type-specific nucleic acid delivery to specific cell lines. Herein, to test the primary-cell-targeting efficiency of the curdlan derivative, we chemically conjugated a macrophage-targeting ligand, mannose, to 6-amino-6-deoxy-curdlan. The resulting curdlan derivative (denoted CMI) readily complexed with siRNA and formed nanoparticles with a diameter of 50-80 nm. The CMI nanoparticles successfully delivered a dye-labeled siRNA to mouse peritoneal macrophages. The delivery efficiency was blocked by mannan, a natural ligand for a macrophage surface mannose receptor (CD206), but not by zymosan, a ligand for the dectin-1 receptor, which is also present on the surface of macrophages. Moreover, CMI nanoparticles were internalized by macrophages only at 37 °C, suggesting that the cellular uptake of CMI nanoparticles was energy-dependent. Furthermore, CMI nanoparticle efficiently delivered siRNA against tumor necrosis factor α (TNFα) to lipopolysaccharide-stimulated primary mouse peritoneal macrophages. In vivo experiments demonstrated that CMI nanoparticles successfully delivered siTNFα to mouse peritoneal macrophages, liver, and lung and induced significant knockdown of the TNFα expression at both messenger RNA and protein levels. Therefore, our design of CMI may be a promising siRNA carrier for targeting CD206-expressing primary cells such as macrophage and dendritic cells.

Sugar Functionalized Synergistic Dendrimers for Biocompatible Delivery of Nucleic Acid Therapeutics.

Sugars containing cationic polymers are potential carriers for in vitro and in vivo nucleic acid delivery. Monosaccharides such as glucose and galactose have been chemically conjugated to various materials of synergistic poly-lysine dendrimer systems for efficient and biocompatible delivery of short interfering RNA (siRNA). The synergistic dendrimers, which contain lipid conjugated glucose terminalized lysine dendrimers, have significantly lower adverse impact on cells while maintaining efficient cellular entry. Moreover, the synergistic dendrimers complexed to siRNA induced RNA interference (RNAi) in the cells and profoundly knocked down green fluorescence protein (GFP) as well as the endogenously expressing disease related gene Plk1. The new synergic dendrimers may be promising system for biocompatible and efficient siRNA delivery.

Synthesis of amphoteric curdlan derivatives for delivery of therapeutic nucleic acids.

Cationic polymers are powerful carriers for intracellular delivery of therapeutic nucleic acids. However, the positively charged macromolecules have considerable cytotoxicity and often induce irreversible damages to the cells and tissues, which greatly negate the clinical application of such materials as drug delivery system. Herein, we report the synthesis of novel amphoteric polymers based on curdlan, and the evaluation of their cytotoxicity as well as the nucleic acid delivery efficiency. ß-(1,3)-polyglucuronic acid, a TEMPO-oxidized derivative of curdlan, was chemically modified by conjugation of tetraethylenepentamine. The resulting amphoteric polymers, denoted tetraethylenepentamine-curdlan (TEPAC) polymers have the degree of substitution (DS) ranging from 25% to 48%. The result of MTT assay indicated that TEPAC polymers have negligible cytotoxicity on HeLa cells and A549 cells. The novel amphoteric polymers efficiently bound with plasmid DNA and delivered pcDNA-eGFP plasmid to 293T cells and induced expression of GFP 48h after the transfection. Moreover, TEPAC polymers delivered siRNA to HeLa cells and HepG2 cells in high efficiency, and induced significant RNAi for the expression of an endogenous gene. Collectively, our data demonstrate that the novel curdlan-based amphoteric polymers are biocompatible and may provide a highly efficient system for the delivery of therapeutic nucleic acids.

Preparation of novel curdlan nanoparticles for intracellular siRNA delivery.

RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70-90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of GFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs.