Polyarginine-Mediated siRNA Delivery: A Mechanistic Study of Intracellular Trafficking of PCL-R15/siRNA Nanoplexes.

As a cell-penetrating peptide, polyarginine is widely used in drug delivery systems based on its membrane permeation ability. Previously, we developed the mPEG-PLA-b-polyarginine(R15) triblock copolymer, which exhibited a high siRNA delivery efficiency both in vitro and in vivo. As a continued effort, here the amphiphilic diblock polymer PCL-R15 was synthesized as a simplified model to further elucidate the structure-activity relationship of arginine-based amphiphilic polymers as siRNA delivery systems, and the cellular trafficking mechanisms of the PCL-R15/siRNA nanoplexes were investigated to understand the interaction patterns between the nanoplexes and cells. Compared to the R15/siRNA complexes, the introduction of PCL moiety was found to result in the stronger interactions with cells and the enhanced transfection efficiency after the formation of condensed nanoplexes. Caveolae-mediated endocytosis and clathrin-mediated endocytosis were major routes for the internalization of PCL-R15/siRNA nanoplexes. The intracellular release of siRNA from nanoplexes was confirmed by fluorescence resonance energy transfer assay. It was also noticed that the internalized PCL-R15/siRNA nanoplexes were transported through digestive routes and trapped in lysosomes, which may be the bottleneck for efficient siRNA delivery of PCL-R15/siRNA nanoplexes. This study investigated the relationship between the polymer structure of PCL-R15 and the cellular interaction patterns, which may render implications on the rational design of polyarginine-based siRNA delivery systems.

Non-viral vectors for RNA delivery.

RNA-based therapy is a promising and potential strategy for disease treatment by introducing exogenous nucleic acids such as messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides (ASO) to modulate gene expression in specific cells. It is exciting that mRNA encoding the spike protein of COVID-19 (coronavirus disease 2019) delivered by lipid nanoparticles (LNPs) exhibits the efficient protection of lungs infection against the virus. In this review, we introduce the biological barriers to RNA delivery in vivo and discuss recent advances in non-viral delivery systems, such as lipid-based nanoparticles, polymeric nanoparticles, N-acetylgalactosamine (GalNAc)-siRNA conjugate, and biomimetic nanovectors, which can protect RNAs against degradation by ribonucleases, accumulate in specific tissue, facilitate cell internalization, and allow for the controlled release of the encapsulated therapeutics.

Enhanced cancer therapeutic efficiency of NO combined with siRNA by caspase-3 responsive polymers.

The combination of nitric oxide (NO) and siRNA is highly desirable for cancer therapy. Here, the furoxans-grafted PEI polymer (FDP) with caspase-3 responsive cleavable DEVD linker was synthesized, and used to bind siRNAs via electrostatic interaction and self-assembled into FDP/siRNA nanoplexes by hydrophobic force. After cellular uptake and lysosomal escape, the FDP/siRNA nanoplexes could achieve GSH-triggered NO release, and then increase the activity of caspase-3. The activated caspase-3 could specifically cleave the DEVD peptide sequence and enhance cell apoptosis. With the cleavage of DEVD peptide sequence, the disassembly of FDP/siRNA nanoplexes was further promoted, thereby resulting in increased siRNAs of ~40% were released at 48 h compared with the caspase-3 non-responsive FDnP/siRNA nanoplexes. By this way, cell apoptosis promotion and cell proliferation inhibition was achieved by siRNA-based downregulation of EGFR protein and the upregulated activity of caspase-3, followed by the enhanced cascade release of NO from FDP/siRNA nanoplexes. Furthermore, in vivo results demonstrated the improved anti-cancer efficiency of FDP/siEGFR nanoplexes without any detectable side effects. Therefore, it is believed that the caspase-3 responsive cleavable furoxans-grafted PEI polymers could provide a potential and efficient enhancement for cancer therapeutic efficiency by the co-delivery of nitric oxide and siRNA.

Effects of pH-sensitive nanoparticles prepared with different polymers on the distribution, adhesion and transition of Rhodamine 6G in the gut of rats.

To investigate the effect of different enteric polymers on the characteristics of pH-sensitive nanoparticles, Rhodamine 6G (Rho) was incorporated in various pH-sensitive nanoparticles. The different patterns of pH-dependent release profiles were observed, although some polymers have the same dissolving pH. The distribution, adhesion and transition of different nanoparticles in rat gut showed significant difference, closely related to the release characteristics of nanoparticles, and their release behaviour are dependent on the dissolving pH and the structure of the polymers, as well as the drug property.Most nanoparticle formulations decreased the distribution and adhesion of Rho in the stomach but increased these values in the intestine. The nanocarriers also control the drug release sites and release rate in the GI tract. In conclusion, pH-sensitive nanoparticles seem favourable for drug absorption and it is important to choose the proper materials to obtain the suitable characteristics for the oral pH-sensitive nanoparticles.

Microfluidic-Based Holonomic Constraints of siRNA in the Kernel of Lipid/Polymer Hybrid Nanoassemblies for Improving Stable and Safe In Vivo Delivery.

A safe and efficient delivery system is critical for clinical application of siRNA. However, the conventional electrostatic interaction-based siRNA nanoplexes with bulk mixing preparation were always unsatisfactory for its stability and safety. In this study, the new core-shell lipid/PCL-PEI/siRNA nanoparticles (LPS NPs) endowing holonomic constraint of siRNA in the inner core were prepared by microfluidic technology. On the microfluidic chip, siRNAs were completely compressed into the inner hydrophilic core of reverse PCL-PEI micelles at a low N/P ratio of 5, followed by coating a neutral lipid membrane to form core-shell nanoparticles, which had a uniform size (120.2 ± 1.4 nm) and a negative charge (-8.8 ± 1.6 mV). Compared to bulk mixing-based LMS NPs, the lower usage of cationic PCL-PEI materials and stronger protection of siRNA in serum were found in the microfluidic-based LPS NPs. Furthermore, it was demonstrated that the LPS NPs exhibited significant downregulation of EGFR mRNA and protein expression level both in vitro and in vivo, and showed significant inhibition of tumor growth following systemic administration along with no obvious systemic toxicity. These findings demonstrated that the microfluidic-based lipid/polymer hybrid nanoassemblies would offer a promising siRNA delivery system for clinical application.

Improved Cell Transfection of siRNA by pH-Responsive Nanomicelles Self-Assembled with mPEG- b-PHis- b-PEI Copolymers.

Here, the novel pH-responsive nanomicelles self-assembled with amphipathic meo-poly(ethylene glycol)- b-poly(l-histidine)- b-polyethylenimine (mPEG- b-PHis- b-PEI, EHE) copolymers based on hydrophobic interaction of PHis with deprotonation of imidazoles were developed for siRNA transfection. The cationic nanomicelles could electrostatically compact siRNA into stable EHE/siRNA nanoplexes with a hydrodynamic diameter of ∼190 nm and present a low toxicity in normal physiological condition (pH ∼ 7.4). Different from pH-irresponsive ECE/siRNA nanoplexes based on mPEG- b-poly(ε-caprolactone)- b-PEI (ECE), the EHE/siRNA nanoplexes exhibited a higher cellular uptake along with an increased ζ-potential (from +18 to +32 mV) when the pH changed from 7.4 to 6.8 (extracellular acidic microenvironments). After cell internalization, the EHE/siRNA nanoplexes also exhibited an enhanced nanostructural disassembling and release of siRNA from lysosomal acidic microenvironments (pH ∼ 5.5). Furthermore, it was demonstrated that the EHE/siEGFR nanoplexes downregulated the expression levels of the corresponding mRNA and protein more efficiently than ECE/siEGFR in HeLa cells. The improved siRNA silencing effects of EHE/siEGFR nanoplexes resulted from the higher cellular uptake and enhanced endosomal/lysosomal escape, which is associated with the pH-responsive disassembly of nanostructure as well as the synergistic "proton sponge" effects of PHis and PEI in EHE copolymers. Therefore, the pH-responsive EHE nanomicelles would be promising and potential carriers for cell transfection of siRNA.

Controlled delivery of recombinant hirudin based on thermo-sensitive Pluronic F127 hydrogel for subcutaneous administration: In vitro and in vivo characterization.

Here we investigated thermo-sensitive Pluronic(R) F127 (PF127) hydrogel for the controlled release of peptide and protein drugs after subcutaneous injection, using an antithrombotic polypeptide, recombinant hirudin variant-2 (rHV2), as the model drug. The in vitro release experiment performed with a membrane-less model at 37 degrees C showed that the release of antithrombotic activity of rHV2 from PF127 gel followed zero-order kinetics and correlated well with the weight percentage of PF127 dissolved, indicating a dissolution-controlled release mechanism. The in vivo result obtained after subcutaneous injection of rHV2-loaded PF127 gel in normal rats demonstrated that PF127 gel improved the bioavailability, prolonged the antithrombotic effect of rHV2, and induced detectable plasma rHV2 concentration for a longer time in comparison with rHV2 aqueous solution. Differential scanning calorimetry, dynamic light scattering and Fourier transform infrared spectroscopy provided evidence of the interaction between PF127 and rHV2, but such interaction was unlikely to interfere the feasibility of this drug delivery system. Our current in vitro and in vivo study suggested that PF127 gel may be useful as an injectable delivery vehicle for peptides and proteins with short half-lives to prolong their therapeutic effect, increase their bioavailability and improve the clinic outcome.

A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia.

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amlodipine, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca2+ stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.

Pharmacokinetics of intravenously administered stealth liposomal doxorubicin modulated with verapamil in rats.

Treatment of cancer through co-administration of anticancer drugs and multidrug resistance (MDR) modulators as a strategy to overcome drug resistance has been extensively explored. However, success has been limited by pharmacokinetic interactions because of non-specific blockade of P-glycoprotein (P-gp) in normal tissues or inability to reach relevant concentrations clinically. We hypothesized that stealth liposomal co-encapsulation of doxorubicin (DOX) with a P-glycoprotein inhibitor, verapamil (DARSLs), may overcome these limitations. Using intravenous (i.v.) administrations, the effects of verapamil (VER) either free (FV) or liposome co-encapsulated with DOX (DARSLs) on the pharmacokinetics and tissue distribution characteristics of DOX either as free (FD) or liposome-encapsulated (LD) were evaluated in normal rats. FV increased (P<0.05) the plasma AUC of free DOX (FD). Preparations containing LD had significant prolonged systemic exposure and slow tissue distribution of DOX. LDFV (liposomal DOX with free verapamil) and DARSLs shared similar DOX pharmacokinetics but the latter showed slower DOX distribution in most tissues studied and slower (P<0.05) DOX biliary transport. The addition of VER into LD in these two preparations significantly increased the AUC (P<0.01) and reduced the clearance (P<0.01) of DOX when compared to LD. Specifically, DARSLs reduced initial DOX distribution to the heart (P<0.05) corresponding to initial alleviation (P<0.05) of bradycardia when compared to other DOX with VER preparations. In conclusion, liposomal co-encapsulation of DOX with VER has promise of significant therapeutic advantages, and should be explored further in therapeutic studies with animal tumor xenograft models.

Preparation and characterization of insulin nanoparticles employing chitosan and poly(methylmethacrylate/methylmethacrylic acid) copolymer.

As most of polypeptides are marginally stable, a mild formulation procedure would be beneficial for the activities of these drugs. The objective of the present study was to develop a novel pH-sensitive nanoparticle system that was suitable for entrapment of hydrophilic insulin but without affecting its conformation. Chitosan was incorporated as a positively charged material, and one of the three poly(methylmethacrylate/methylmethacrylic acid) copolymers, consisting of Eudragit L100-55, L100, and S100, was used as a negatively charged polymer for preparation of three insulin nanoparticles, respectively. Three nanoparticles obtained were spherical. The mean diameters were in the range from 200 nm to 250 nm, and the entrapment efficiencies, from 50% to 70%. The surface analysis indicated that insulin was evenly distributed in the nanoparticles. Polymer ratio of chitosan to Eudragit was the factor which influenced the nanoparticles significantly. Characterization results showed that the electrostatic interactions existed, thus providing a mild formulation procedure which did not affect the chemical integrity and the conformation of insulin. In vitro release studies revealed that all three types of the nanoparticles exhibited a pH-dependant characteristic. The modeling data indicated that the release kinetics of insulin was nonlinear, and during the release process, the nanoparticles showed a polynomial swelling. On overall estimation, the insulin chitosan-Eudragit L100-55 nanoparticles may be better for the oral delivery. This new pH-sensitive nanoparticle formulation using chitosan and Eudragit L100-55 polymer may provide a useful approach for entrapment of hydrophilic polypeptides without affecting their conformation.

Thymopentin-loaded pH-sensitive chitosan nanoparticles for oral administration: preparation, characterization, and pharmacodynamics.

Thymopentin, a potent immunomodulating drug, was incorporated into pH-sensitive chitosan nanoparticles prepared by ionic gelation of chitosan with tripolyphosphate anions and then coated with Eudragit S100 to improve the stability and the oral bioavailability. Nanoparticles particle size and zeta potential were measured by photo correction spectroscopy and laser Dopper anemometry. Its morphology was examined by environment scan electron microscope. The encapsulation efficiency and the release in vitro were determined by HPLC. Enzymatic stabilization was expressed by the enzymatic degradation of aminopeptidase. Biological activity of TP5 loaded in nanoparticles was assayed by lymphocyte proliferation test in vitro and the immune function (CD4+/CD8+) of irradiated rat in vivo. The results obtained demonstrated that the average sizes of pH-sensitive chitosan nanoparticles were 175.6 +/- 17 nm, the zeta potential was 28.44 +/- 0.5 mV and the encapsulation efficiency was 76.70 +/- 2.6%. The cumulative release percentages of thymopentin from the pH-sensitive nanoparticles were 24.65%, 41.01%, and 81.44% incubated in different medium, 0.1 N HCl, pH 5.0 PBS, and pH 7.4 PBS, respectively. The pH-sensitive chitosan nanoparticles could efficiently protect TP5 from enzymatic degradation and prolong the degradation half-time of TP5 from 1.5 min to 15 min. It was demonstrated from the lymphocyte proliferation test that the nanoparticle-encapsulated TP5 still kept its biological activity. In immunosuppression rats, the lowered T-lymphocyte subsets values were significantly increased and the raised CD4+/CD8+ ratio was evidently reduced. These results indicated that pH-sensitive chitosan nanoparticles may be used as the vector in oral drug delivery system for TP5.

Long-term studies on the stability and oral bioavailability of cyclosporine A nanoparticle colloid.

The present study was geared at the long-term stability and the changes in oral bioavailability of CyA Eudragit S100 nanoparticles stabilized by suspending agents. CyA Eudragit S100 nanoparticle colloids were prepared by quasi-emulsion solvent diffusion technique and they were mixed with Xanthan gum to obtain suspended nanoparticle colloids. The suspended nanoparticle colloids were preserved at different temperatures for different period of time, as long as 18 months. During the storage period, the CyA concentration, particle size, pH and viscosity were determined. The results indicated that CyA concentration, particle size and viscosity of the colloids had no obvious change. However, the pH increased slightly from 5.5 to about 6.4. The results of bioavailability and pharmacokinetic study revealed that all formulations of nanoparticles showed higher C(max) and higher AUC(0-24) values than that of reference (Neoral). The relative bioavailability of S-CyA-S100 NP initial compared with Neoral was 162.8%. The C(max) and AUC(0-24) values of nanoparticle formulations at 12 and 18 months were both lower than that of the initial. The bioequivalency was suggested between the tested nanoparticle formulations at the initial and 12 months. It was deduced by surface analysis, TEM observation, in vitro release as well as the characteristics of Eudragit S100 that the decrease in bioavailability might be due to the pH change of the nanoparticle colloid.

Therapeutic and toxicological evaluations of cyclosporine a microspheres as a treatment vehicle for uveitis in rabbits.

AIM: This study was undertaken to investigate the therapeutic efficacy and the toxicity of the intravitreal biodegradable poly(dl-lactide-co-glycolide)co-polymer microspheres containing cyclosporin A (CsA-PLGA-MS) on experimental uveitis in rabbits. METHODS: CsA-PLGA-MS that had been prepared by a solvent evaporation approach were characterized for morphology, particle size, entrapment efficiency, and in vitro release profile of CsA-PLGA-MS. Therapeutic efficacy of the CsA-PLGA-MS was evaluated by scoring of the inflammation, aqueous leukocyte counting, aqueous protein determination, and histological examination in the experimental rabbits with artificial uveitis induced by the injection of lipopolysaccharide. The toxicity was investigated by slit-lamp examination, indirect ophthalmoscopy, and electroretinography (ERG) in the noninflamed rabbit eye. RESULTS: The CsA-PLGA-MS were spherical in shape, with an average particle size of nearly 50 microm and an entrapment efficiency of more than 80%. The compositions of the formulation that was most effective in the in vivo studies included CsA, PLGA, and 3% Pluronic F68. In vitro released cyclosporine A from the optimized microspheres was approximately 25% during the 60-day incubation at 37 degrees C. It was demonstrated that the intravitreal injection of the optimized CsA-PLGA-MS decreased significantly the severity of the inflammatory signs, cellular infiltrate, aqueous leukocyte counts, and protein levels in the eyes of experimental rabbits with uveitis, compared to other formulations. Also, the preparation did not cause obvious toxicity in the noninflamed eyes of rabbits, except that the ERG b-wave amplitude for the test eyes was reversibly depressed, compared to those of the control eyes at 2 weeks, which almost recovered at the end of 6 weeks. CONCLUSIONS: The CsA-PLGA-MS preparation might be useful in the treatment of patients with severe chronic posterior uveitis who cannot tolerate systemic or periocular therapy.

[Effects of absorption enhancers on intestinal absorption of lumbrokinase].

AIM: To explore the intestinal absorption characteristics of lumbrokinase (YJM-I) in the absence or presence of various absorption enhancers and to find the optimum intestinal site for YJM-I absorption. METHODS: The absorption kinetics and absorption intestinal sites for YJM-I absorption were investigated with the method of diffusion cell in vitro, duodenum bolus injection, recirculating perfusion and in situ duodenum perfusion in vivo. RESULTS: YJM-I could be transported into blood and kept its biological activity across intestinal endothelial membrane after administration via duodenum site, whereas with lower bioavailability. Some of the absorption enhancers were shown good enhancement effects on intestinal absorption of YJM-I in vitro and in situ experiments. The order of enhanced efficiencies of various enhancers on duodenum, ileum and jejunum in vitro permeation experiments were shown as follows: 1% chitosan > 1% SDCh > 1% Na2EDTA > 1% SDS > 1% sodium caprylate > 1% poloxamer > 1% HP-beta-CD. The order of enhanced efficiencies of various enhancers on duodenum absorption of YJM-I in vivo were as follows: 2.5% SDCh > 2.5% Na2EDTA > 2.0% chitosan > 2.5% SDS > 2.5% sodium caprylate > 2.5% Poloxamer > 2.5% HP-beta-CD. CONCLUSION: The results indicated that the absorption of YJM-I could be enhanced by various enhancers, and duodenum was the optimum absorption site of YJM-I. Furthermore, bio-adhesive chitosan might be a potential enhancer of intestinal YJM-I absorption.

Systemic delivery of siRNA by T7 peptide modified core-shell nanoparticles for targeted therapy of breast cancer.

Systemic delivery of siRNA is the most challenging step to transfer RNAi to clinical application for breast cancer therapy. In this study, the tumor targeted, T7 peptide modified core-shell nanoparticles (named as T7-LPC/siRNA NPs) were constructed to achieve effective systemic delivery of siRNA. The core-shell structure of T7-LPC/siRNA NPs enables them to encapsulate siRNA in the core and protect it from RNase degradation during circulation. In vitro cellular uptake and gene silencing experiments demonstrated that T7-LPC/siEGFR NPs could deliver EGFR siRNA into breast cancer cells through receptor mediated endocytosis and effectively down-regulate the EGFR expression. In vivo distribution study proved the T7-LPC/siRNA NPs could deliver fluorescence labeled siRNA to the tumor site more efficiently than the non-targeted PEG-LPC/siRNA NPs after intravenous administration. Furthermore, the experiments of in vivo tumor therapy confirmed that intravenous administration of T7-LPC/siEGFR NPs led to an effective EGFR down-regulation and an obvious inhibition of breast tumor growth, with little activation of immune responses and negligible body weight loss. These results suggested that T7-LPC/siRNA NPs could be an effective and safe systemic siRNA delivery system for RNAi-based breast cancer therapy.

[The in vitro cytotoxicity and in vivo toxicity of doxorubicin antiresistant stealth liposomes].

AIM: Multidrug resistance ( MDR) as a major obstacle to successful clinical cancer chemotherapy, searching a novel effective antiresistant drug would be necessary. METHODS: A novel doxorubicin anti-resistant stealth liposomes (DARSLs) was prepared by co-encapsulating doxorubicin (DOX) and verapamil (VER) into stealth liposomes with ammonium sulfate gradient remote loading approach. In vitro cytotoxity of various DOX formulations and in vivo toxicity of DARSLs were evaluated using DOX-resistant rat prostate cancer cell line (MLLB2), human uterus sarcoma cell line (MES-SA/DX5) and normal SD rats, separately. RESULTS: The DARSLs liposome suspensions mainly consisted of homogeneous large unilamellar vesicles (LUV) with average particle size of (118.1 +/- 22.3) nm. Encapsulation efficiencies of DOX and VER in DARSLs were more than 90% and about 70%, respectively, when the ratio of DOX/VER/Lipid was 1: 0.11 :10 (w/w/w). In vitro cytotoxicity tests of the DARSLs using rat prostate cancer cell line (MLLB2) and human uterus sarcoma cell line (MES-SA/DX5) showed that 5 micromol x L(-1) VER significantly reversed DOX-resistance of these 2 cell lines and DARSLs was the most effective on inhibition of DOX-resistant cell growth. Besides, compared to FDFV, much slower DOX distribution (confocal microscopy) to nuclei and cytoplasm in MLLB2 cells for DARSLs suggested that it might possess distinct mechanism of cytotoxicity. Systemic and cardiac toxicity evaluations in normal SD rats suggested that liposomal encapsulation could significantly improve the severe cardiotoxicity arising from simultanous administration of DOX and VER. CONCLUSION: DARSLs is a novel anticancer liposome formulation with lower cardiotoxicity, effective drug-resistance reversal and intravenous injection.

Core-shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery.

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core-shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.

Self-assembly cationic nanoparticles based on cholesterol-grafted bioreducible poly(amidoamine) for siRNA delivery.

In this study, a series of bioreducible poly(amidoamine)s grafting different percentages of cholesterol (rPAA-Ch14: 14%, rPAA-Ch29: 29%, rPAA-Ch57: 57% and rPAA-Ch87: 87%) was synthesized and used for siRNA delivery. These amphiphilic polymers were able to self-assemble into cationic nanoparticles in aqueous solution at low concentrations. The nanoparticle formation was evidenced via cryo-transmission electron microscope (Cryo-TEM) and dynamic light scattering analysis. The average hydrodynamic size of rPAA-Ch blank nanoparticles was about 80-160 nm with zeta potential of 50-60 mV. Also, the effects of different percentages of cholesterol grafted onto rPAA on physicochemical characteristics, in vitro cytotoxicity, cellular uptake, VEGF gene silencing efficacy and translocation mechanism of rPAA-Ch/siRNA complexes were investigated. The results showed that rPAA-Ch57 polymer was not only able to form stable nanocomplexes and possess high cell uptake, but also to exhibit the best in vitro VEGF gene silencing efficacy and the best in vivo tumor growth inhibition effect when it was formulated with VEGF-siRNA. Moreover, the observations of confocal laser scanning microscope (CLSM) and the study of cholesterol competitive inhibition demonstrated that endosomal/lysosomal escape and cytoplasmic dissociation of rPAA-Ch57/siRNA complexes were dependent on the "proton sponge effect" and disulfide cleavage, following internalization with cholesterol-related endocytosis pathway and subsequent transportion into endosomes/lysosomes. These findings indicated that the rPAA-Ch57 polymer should be a promising and potent carrier for siRNA delivery.

Self-assembly nanomicelles based on cationic mPEG-PLA-b-Polyarginine(R15) triblock copolymer for siRNA delivery.

Due to the absence of safe and effective carriers for in vivo delivery, the applications of small interference RNA (siRNA) in clinic for therapeutic purposes have been limited. In this study, a biodegradable amphiphilic tri-block copolymer (mPEG(2000)-PLA(3000)-b-R(15)) composed of monomethoxy poly(ethylene glycol), poly(d,l-lactide) and polyarginine was synthesized and further self-assembled to cationic polymeric nanomicelles for in vivo siRNA delivery, with an average diameter of 54.30 ± 3.48 nm and a zeta potential of approximately 34.8 ± 1.77 mV. The chemical structures of the copolymers were well characterized by (1)H NMR spectroscopy and FT-IR spectra. In vitro cytotoxicity and hemolysis assays demonstrated that the polymeric nanomicelles showed greater cell viability and haemocompatibility than those of polyethyleneimine (PEI) or R(15) peptide. In vitro experiments demonstrated that EGFR targeted siRNA formulated in micelleplexes exhibited approximately 65% inhibition of EGFR expression on MCF-7 cells in a sequence-specific manner, which was comparable to Lipofectamine™ 2000. The results of intravenous administration showed Micelleplex/EGFR-siRNA significantly inhibited tumor growth in nude mice xenografted MCF-7 tumors, with a remarkable inhibition of EGFR expression. Furthermore, no positive activation of the innate immune responses and no significant body weight loss was observed during treatment suggested that this polymeric micelle delivery system is non-toxic. In conclusion, the present nanomicelles based on cationic mPEG(2000)-PLA(3000)-b-R(15) copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA.

A lipid nanoparticle system improves siRNA efficacy in RPE cells and a laser-induced murine CNV model.

PURPOSE: To explore the possibility of the PEGylated liposome-protamine-hyaluronic acid nanoparticles (PEG-LPH-NP) loaded with siRNA (PEG-LPH-NP-S) in ARPE19 cells and a laser-induced rat model for the treatment of choroidal neovascularization (CNV). METHODS: PEG-LPH-NP-S was characterized by dynamic light scattering and transmission electron microscopy (TEM). The encapsulation efficiency of siRNA in PEG-LPH-NP was analyzed by ultracentrifugation, whereas the protection of siRNA by PEG-LPH-NP was evaluated by electrophoresis. Human RPE cells (ARPE19) were used as the cell model for the studies of cellular uptake and the inhibition of VEGFR1 expression, visualized by a laser scanning confocal microscope. The area of CNV in the laser-induced rat model after intravitreous injection was measured. The distribution of the lipid nanoparticles in the retina after intravitreous administration was investigated by fluorescence microscopy. Finally, the TUNEL test and morphologic observation of the retina were conducted. RESULTS: It was indicated that PEG-LPH-NP-S was approximately 132 nm in particle size with a positive charge of approximately 20 mV, whereas the encapsulation efficiency of siRNA in PEG-LPH-NP was >95%. PEG-LPH-NP could protect the siRNA load and could facilitate the intracellular delivery of fluorescein-labeled siRNA to ARPE19 cells. VEGFR1 expression in ARPE19 cells could be inhibited, and the CNV area in the murine model could be reduced more effectively by PEG-LPH-NP-S compared with naked siRNA and by PEG-LPH-NP with negative siRNA. It seems that the toxicity of PEG-LPH-NP-S on the rat retina is low, based on the results of TUNEL testing and morphologic observation. CONCLUSIONS: PEG-LPH-NP may be a promising lipid nanoparticle system for the siRNA treatment of CNV.