siRNA Encapsulation Efficiency Research Articles

Enhancing Gene Therapy through Ultradeformable Vesicles for Efficient siRNA Delivery.

Gene therapy is a revolutionary approach aimed at treating various diseases by manipulating the expression of specific genes. The composition and formulation of ultra-deformable vesicles play a crucial role in determining their properties and performance as siRNA delivery vectors. In the development of ultra-deformable vesicles for siRNA delivery, careful lipid selection and optimization are crucial for achieving desirable vesicle characteristics and efficient siRNA encapsulation and delivery. The stratum corneum acts as a protective barrier, limiting the penetration of molecules, including siRNA, into the deeper layers of the skin. Ultradeformable vesicles offer a promising solution to overcome this barrier and facilitate efficient siRNA delivery to target cells in the skin. The stratum corneum, the outermost layer of the skin, acts as a significant barrier to the penetration of siRNA.These engineering approaches enable the production of uniform and well-defined vesicles with enhanced deformability and improved siRNA encapsulation efficiency. Looking ahead, advancements in ultra-deformable vesicle design and optimization, along with continued exploration of combination strategies and regulatory frameworks, will further drive the field of ultra-deformable vesicle-based siRNA delivery.

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends.

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI-PCL) (800Da-40kDa) and PEI-PCL-PEI (5-5-5kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG-PCL) (5kDa-4kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100nm. PEG-PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI-PCL-PEI and PEG-PCL show higher siRNA encapsulation efficiency than PEI-PCL/PEG-PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI-PCL-PEI/PEG-PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.

Cell-free protein synthesis of influenza virus hemagglutinin HA2-integrated virosomes for siRNA delivery

It is well known that the difficulty of siRNA therapeutic application is the lack of safe and effective delivery vector. Virosome is a nano vesicle composed of lipid membrane and membrane protein. It retains fusion protein without virus genetic material, and therefore has the reduced immunogenicity compared with viral vector. Virosomes have the potential to deliver protein and nucleic acid drugs, but the traditional preparation method of virosomes is quite limited. In this study, we firstly proposed to synthesize influenza virus hemagglutinin HA2 virosomes by cell-free protein synthesis. In this study, liposomes provided the hydrophobic lipid bilayer environment for the formation of HA2 protein multimer, which inhibited the aggregation of hydrophobic HA2 and improved HA2 protein expression. Chitosan as a rigid core adsorbed siRNA and improved the encapsulation efficiency of siRNA. In conclusion, the cell-free protein synthesis was used to prepare HA2 virosomes, which paves the way for constructing a novel nano vector with high delivery efficiency and biosafety for the delivery of siRNA.

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Designing siRNA/chitosan-methacrylate complex nanolipogel for prolonged gene silencing effects

Despite immense revolutionary therapeutics potential, sustaining release of active small interfering RNA (siRNA) remains an arduous challenge. The development of nanoparticles with siRNA sustained release capabilities provides an avenue to enhance the therapeutic efficacy of gene-based therapy. Herein, we present a new system based on the encapsulation of siRNA/chitosan-methacrylate (CMA) complexes into liposomes to form UV crosslinkable Nanolipogels (NLGs) with sustained siRNA-release properties in vitro. We demonstrated that the CMA nanogel in NLGs can enhance the encapsulation efficiency of siRNA and provide sustained release of siRNA up to 28 days. To understand the particle mechanism of cellular entry, multiple endocytic inhibitors have been used to investigate its endocytosis pathways. The study saw positively charged NLGs entering cells via multiple endocytosis pathways, facilitating endosomal escape and slowly releasing siRNA into the cytoplasm. Transfection experiments confirmed that the crosslinked NLG delivery system provides effective transfection and prolonged silencing effect up to 14 days in cell cultures. We expect that this sustained-release siRNA NLG platform would be of interest in both fundamental biological studies and in clinical applications to extend the use of siRNA-based therapies.

SiRNA Nanohybrid Systems: False Hope Or Feasible Answer in Cancer Management

Finding out predisposition and makeup alterations in cancer cells has prompted the exploration of exogenous small interference RNA (siRNA) as a therapeutic agent to deal with cancer. siRNA is subjected to many limitations that hinderits cellular uptake. Various nanocarriers have been loaded with siRNA to improve their cellular transportation and have moved to clinical trials. However, many restrictions as low encapsulation efficiency, nanocarrier cytotoxicity and premature release of siRNA have impeded the single nanocarrier use. The realm of nanohybrid systems has emerged to overcome these limitations and to synergize the criteria of two or more nanocarriers. Different nanohybrid systems that were developed as cellular pathfinders for the exogenous siRNA to target cancer will be illustrated in this review.

Abstract 306: Cationic cholesterol liposomes for combination therapy to treat drug-resistant ovarian cancer

Abstract Introduction: Ovarian cancer is the fifth leading cause of cancer mortality in women, with nearly 75% of women who respond to initial platinum-based chemotherapy experiencing relapse due to drug resistance [1,2]. Liposomes are a promising solution to overcoming drug resistance due to their biocompatibility and capacity to encapsulate hydrophilic and hydrophobic drugs as well as complex small interfering RNA (siRNA), which have the potential to downregulate the expression of genes related to drug resistance [3]. We aim to synthesize and characterize a cationic liposomal system to deliver siRNA and paclitaxel (PTX) to ovarian cancer cells. Methods: Cholesterol (CHOL) liposomes were synthesized by the thin-film hydration method. Dynamic light scattering (DLS) was used to determine the size, polydispersity index (PDI), and zeta potential of the liposomes. Uptake of liposomes into OVCAR3 and OVCAR3-T40, a wild-type and a paclitaxel-resistant human adenocarcinoma cell line, was examined using fluorescence microscopy. The cytotoxicity of unloaded CHOL liposomes was evaluated through MTS assay on OVCAR3 and OVCAR3-T40 cells. Results: PTX- and siRNA-loaded CHOL liposomes had an average diameter of 114.9 ± 10.35 nm and a zeta potential of 27.6 ± 1.79 mV. Blank CHOL liposomes had an average diameter of 123.0 ± 2.49 nm and zeta potential of 32.3 ± 2.16 mV. All formulations of liposomes were cationic and formed monodisperse nanoparticles. The encapsulation efficiency of siRNA and PTX was 99.8% and 80.4% respectively. Coumarin 6, a hydrophobic model drug, was loaded into liposomes to verify cellular uptake through fluorescent imaging. Results demonstrated that the liposomal system was efficiently delivered intracellularly. Blank liposomes were used to determine the toxicity of the delivery system. The unloaded liposomes were not cytotoxic to both the wild-type and drug-resistant cell lines at concentrations up to 75 µg/mL, and therefore, cytotoxicity of drug-loaded liposomes can be attributed to paclitaxel, siRNA, or combination treatment. Conclusions: Liposomes were successfully formed with a monodisperse size, exhibited effective drug and siRNA loading, and were internalized into OVCAR3 and OVCAR3-T40 cells. Future work includes investigating the efficacy of the liposomal system in mediating gene silencing. Acknowledgements: This work was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740 and Clemson Creative Inquiry.

Abstract 281: pH-sensitive liposome for siRNA delivery to treat drug-resistant ovarian cancer

Abstract Introduction: With a 5-year survival rate of 47%, ovarian cancer is the 5th leading cause of death amongst women worldwide. Over 75% of patients experience recurrence after initial treatment, indicating a need for improved treatment options. Drug resistance is a major barrier hindering the success of current treatment methods. Our study analyzes the characteristics of a stimuli-sensitive liposomal delivery system for combatting drug resistance. Our delivery system will deliver bioactive siRNAs targeting genes related to drug resistance, cell proliferation, and apoptosis. In this study, we investigate the characteristics of the liposomes to determine particle size, surface charge, and ability to encapsulate/bind both siRNAs. We also begin to investigate the delivery potential of the pH-sensitive liposomal formulation in vitro using ovarian cancer cell lines. Methods: Empty and siRNA loaded cationic, pH-sensitive liposomes (CHEMS-LPs) were synthesized by the thin-film hydration method. Liposome size, zeta potential, and polydispersity index (PDI) were measured by dynamic light scattering (DLS). To measure siRNA encapsulation efficiency, fluorescently labeled siRNA was loaded into CHEMS-LPs and subjected to centrifugation to pellet the LPs. Fluorescence spectroscopy was used to detect siRNA in the supernatant. The toxicity of unloaded CHEMS-LPs was determined by an MTS assay using OVCAR3 (drug-sensitive) and OVCAR3-T40 (drug-resistant) human ovarian cancer cells. Results: The size and zeta potential of blank and siRNA-loaded CHEMS-LPs were 97.88 ± 2.39 nm and 29.0 ± 2.00 mV, and 80.78 ± 0.77​ nm and 13.1 ±1.66​ mV, respectively. The positively charged zeta potential confirms the cationic nature of our liposomes. The PDI demonstrated that the liposomes were unimodal and monodisperse with PDI values of less than 0.300 for each formulation. In addition, siRNA was successfully bound to CHEMS-LPs through electrostatic interaction with the cationic lipid layer, resulting in an encapsulation efficiency of 99.6% Conclusion: CHEMS-LPs are pH-sensitive, cationic, monodisperse liposomes able to encapsulate siRNAs in order to mediate delivery into ovarian cancer cells. Their stable structure, positive charge, and low cytotoxicity is promising for future studies, including delivery of bioactive siRNAs to stimulate downregulation of target genes related to drug resistance. Acknowledgements: This work was supported in part by the National Science Foundation EPSCoR Program under Award # OIA-1655740. We would like to thank George Duran from Stanford University for donating the OVCAR3-T40 cell line. Citation Format: Kharimat Lora Alatise, Samantha Gardner, Angela Alexander-Bryant. pH-sensitive liposome for siRNA delivery to treat drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 281.

Development of lipid nanoparticles for delivery of siRNA to neural cells

Lipid nanoparticles (LNPs) are a clinically approved platform to deliver siRNA to the liver. We are optimizing this platform to deliver siRNA for the treatment of pain. siRNA against TRPV1 (siTRPV1) was chosen as a proof of concept. siTRPV1-LNPs were formulated using C12-200, an ionizable cationic lipid and helper lipids. Their particle size and surface charge was characterized using dynamic light scattering and siRNA encapsulation efficiency was determined using a Ribogreen assay. A panel of breast cancer cell lines (MCF-7, MDA-MB-231 and BT-549) that express TRPV1 were selected for in vitro screening of LNPs to determine their cytocompatibility and gene knockdown effects using an ATP assay, qPCR and western blotting. The effects of mixing speeds during LNP formulation, slow vs. fast, were compared for determining their effects on %siRNA encapsulation. The values increased from 74.4% at slow mixing to 83.8% at faster mixing speeds. siRNA-LNPs showed a particle diameter of ca. 251 nm compared to 434 nm for empty LNPs. A lower polydispersity index of 0.15 was observed for siRNA-LNPs compared to 0.41 for empty LNPs, suggesting narrower particle size distribution upon siRNA loading. Zeta potential of siRNA-LNPs (+0.15 mV) was slightly lower compared to empty LNPs (+0.41 mV). siRNA-LNPs had a negligible impact on cell viability, which was >95% in all three cell lines and primary rat dorsal root ganglion neurons four hours post-exposure. We are currently optimizing transfection parameters to determine TRPV1 knockdown in primary DRG cultures. LNPs were well-tolerated by TRPV1-expressing cell lines and primary DRG cultures, allowing their further exploration for TRPV1 silencing.

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics.

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Peptide/Lipid-Associated Nucleic Acids (PLANAs) as a Multicomponent siRNA Delivery System.

RNAi is a biological process that utilizes small interfering RNA (siRNA) to prevent the translation of mRNA to protein. This mechanism could be beneficial in preventing the overexpression of proteins in cancer. However, the cellular delivery of siRNA has proven to be challenging due to its inherent negative charge and relative instability. Here, we designed a multicomponent delivery system composed of a specifically designed peptide (linear or cyclic fatty acyl peptide conjugates and hybrid cyclic/linear peptides) and several lipids (DOTAP, DOPE, cholesterol, and phosphatidylcholine) to form a nanoparticle, which we have termed as peptide lipid-associated nucleic acids (PLANAs). Five formulations were prepared (a formulation with no peptide, which was named lipid-associated nucleic acid or LANA, and PLANA formulations A-D) using a mini extruder to form uniform nanoparticles around 100 nm in size with a slightly positive charge (less than +10 mv). Formulations were evaluated for peptide incorporation, siRNA encapsulation efficiency, release profile, toxicity, cellular uptake, and protein silencing. Our experiments showed effective encapsulation of siRNA (>95%), a controlled release profile, and negligible toxicity in formulations that did not contain a positively charged lipid. The results also revealed that PLANAs C and D exhibited optimum cellular uptake (with 80-90% siRNA-positive cells for most of the formulations). PLANA D formulation was selected to silence two model proteins (Src and RPS6KA5) in the triple-negative human breast cancer cell line MDA-MB-231, with promising silencing efficiency, which diminished the expression of RPS6KA5 and Src to approximately 29 and 38% compared to naïve cells, respectively. Many approaches have been investigated for safe and efficient delivery of nucleic acids in the last 20 years; however, many have failed due to the multifaceted challenges to overcome. Our results show a promising potential for a multicomponent design that incorporates different components for a variety of delivery tasks, which warrants further investigation of PLANAs in vivo.

DDAB cationic lipid-mPEG, PCL copolymer hybrid nano-carrier synthesis and application for delivery of siRNA targeting IGF-1R into breast cancer cells.

To use siRNA molecule as a therapeutic agent in gene silencing, an efficient delivery system is necessary. Stability and clearance by reticuloendothelial of siRNA still remains the major challenges for clinical application. Herein, we could develop new lipid-polymer hybrid nanoparticles (LPHNP) as a siRNA carrier to silence insulin-like growth factor type I (IGF-1R) gene overexpression in MCF-7 human breast cancer cell line. Dimethyldioctadecylammonium bromide-methoxy poly(ethylene glycol)-poly (ε-caprolactone) (DDAB-mPEG-PCL) LPHNPs were synthesized using a single step nanoprecipitation method and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM) microscope. Cytotoxicity of the nanoparticles was assessed in the MCF7 cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Desired LPHNP-siRNA complex was determined using different Nitrogen:Phosphate ratio (N/P) ratios and gel retardation. To determine the encapsulation efficiency of siRNA (%) in LPHNP, its absorbance was measured. The effect of the siRNA-LPHNP complex on IGF-1R silencing was assessed by reverse transcription-polymerase chain reaction (RT-PCR) RESULTS: LPHNP was synthesized using a single-step sonication method with a size below 100nM. The viability of cells treated with hybrid nanoparticles was significantly greater than the corresponding cationic lipid (P < 0.01). As demonstrated by gel retardation assay, efficient siRNA binding to LPHNP occurred at N/P equal to 40 and siRNA encapsulation efficiency was found to be 95% ± 4 at this ratio. LPHNP-IGF-1R siRNA complex could be able to down-regulate the target more efficiently when it compared with the corresponded controls (P < 0.001). In conclusion, our results suggest that DDAB cationic lipid and mPEG-PCL copolymer hybrid nanoparticle may be a good candidate for efficient siRNA delivery.

Designing siRNA/Chitosan-Methacrylate Complex Nanolipogel for Prolonged Gene Silencing Effects

Despite the great therapeutics potential, sustained release of small interfering RNA (siRNA) remains a challenge. Development of siRNA sustained release nanoparticle provides possibility to further enhance the therapeutic efficacy in gene-based therapy. Herein, we present a new system based on the encapsulation of siRNA/chitosan-methacrylate (CMA) complexes into liposomes to form UV crosslinkable Nanolipogels (NLGs) with sustained siRNA-release properties, both in vitro and inside cells. We demonstrated that the CMA nanogel in NLGs can enhance the encapsulation efficiency of siRNA and provide sustained release of siRNA up to 28 days. To understand the mechanism of cellular uptake, multiple endocytic inhibitors have been used to study the endocytosis pathways. This study proved that positively charged NLGs can enter the cells via multiple endocytosis pathways, facilitating the endosome escape and slowly releasing the siRNA into the cytoplasm. Transfection experiments confirmed that the crosslinked NLG delivery system provides effective transfection and prolonged silencing effect for up to 14 days. We expect that this sustained-release siRNA NLG platform would be of interest in both fundamental biological studies and in clinical applications to extend the use of siRNA-based therapies.

Self-crosslinkable chitosan-hyaluronic acid dialdehyde nanoparticles for CD44-targeted siRNA delivery to treat bladder cancer

Bladder cancer is one of the concerning malignancies worldwide, which is lacking effective targeted therapy. Gene therapy is a potential approach for bladder cancer treatment. While, a safe and effective targeted gene delivery system is urgently needed for prompting the bladder cancer treatment in vivo. In this study, we confirmed that the bladder cancer had CD44 overexpression and small interfering RNAs (siRNA) with high interfere to Bcl2 oncogene were designed and screened. Then hyaluronic acid dialdehyde (HAD) was prepared in an ethanol-water mixture and covalently conjugated to the chitosan nanoparticles (CS-HAD NPs) to achieve CD44 targeted siRNA delivery. The in vitro and in vivo evaluations indicated that the siRNA-loaded CS-HAD NPs (siRNA@CS-HAD NPs) were approximately 100 nm in size, with improved stability, high siRNA encapsulation efficiency and low cytotoxicity. CS-HAD NPs could target to CD44 receptor and deliver the therapeutic siRNA into T24 bladder cancer cells through a ligand-receptor-mediated targeting mechanism and had a specific accumulation capacity in vivo to interfere the targeted oncogene Bcl2 in bladder cancer. Overall, a CD44 targeted gene delivery system based on natural macromolecules was developed for effective bladder cancer treatment, which could be more conducive to clinical application due to its simple preparation and high biological safety.

Development of a Microfluidic-Based Post-Treatment Process for Size-Controlled Lipid Nanoparticles and Application to siRNA Delivery.

Microfluidic methodologies for preparation of lipid nanoparticles (LNPs) based on an organic solvent injection method enable precise size control of the LNPs. After preparation of LNPs, the organic solvent injection method needs some post-treatments, such as overnight dialysis or direct dilution with a buffer solution. LNP production using the microfluidic-based organic solvent injection method is dominated by kinetics rather than thermodynamics. Kinetics of ethanol removal from the inner and outer membranes of LNPs could induce a structural change in the membrane that could lead to fusion of LNPs. However, the effects of microfluidic post-treatment on the final size of LNPs have not been sufficiently understood. Herein, we investigated the effect of the post-treatment processes on the final product size of LNPs in detail. A simple baffle device and a model lipid system composed of a neutral phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and cholesterol were used to produce the LNPs. We demonstrated that flow conditions of the post-treatment diluting the remaining ethanol in the LNP suspension affected the final product size of LNPs. Based on the findings, we developed an integrated baffle device composed of an LNP production region and a post-treatment region for a microfluidic-based LNP production system; this integrated baffle device prevented the undesirable aggregation or fusion of POPC LNPs even for the high-lipid-concentration condition. Finally, we applied our concept to small interfering RNA (siRNA) delivery and confirmed that no significant effects due to the continuous process occurred on the siRNA encapsulation efficiency, biological distribution, and knockdown activity. The microfluidic post-treatment method is expected to contribute to the production of LNPs for practical applications and the development of novel LNP-based nanomedicines.

Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells.

RNA interference (RNAi) offers the potential to selectively silence disease-related genes in defined cell subsets. Translation into the clinical routine is, however, still hampered by the lack of efficient carrier systems for therapeutic siRNA, endosomal entrapment presenting a major hurdle. A promising siRNA delivery system has previously been developed on the base of polyethylenimine (PEI) and the targeting ligand transferrin (Tf) to specifically reach activated T cells in the lung. In the present work, the focus is on optimizing Tf-PEI polyplexes for gene knockdown in primary activated T cells by improving their endosomal escape properties. Blending of the conjugate with membrane lytic melittin significantly enhanced endosomal release and thereby cytoplasmic delivery, while maintaining selective T cell targeting abilities and overall cell tolerability. The gathered data furthermore demonstrate that melittin addition also distinctly improves several other essential particle characteristics, such as siRNA encapsulation efficiency and stability in lung lining fluids. In conclusion, this results in a novel upgraded siRNA delivery system that is not only able to specifically deliver its payload to the desired target cells via receptor-mediated endocytosis, but also shows enhanced release from endosomal vesicles in order to initiate RNAi in the cytoplasm.