High Transfection Efficiency Research Articles

Arginine-Rich Cell-Penetrating Peptide-Mediated Transduction of Mouse Nasal Cells with FOXP3 Protein Alleviates Allergic Rhinitis.

Intranasal corticosteroids are effective medications against allergic rhinitis (AR). However, mucociliary clearance promptly eliminates these drugs from the nasal cavity and delays their onset of action. Therefore, a faster, longer-lasting therapeutic effect on the nasal mucosa is required to enhance the efficacy of AR management. Our previous study showed that polyarginine, a cell-penetrating peptide, can deliver cargo to nasal cells; moreover, polyarginine-mediated cell-nonspecific protein transduction into the nasal epithelium exhibited high transfection efficiency with minimal cytotoxicity. In this study, poly-arginine-fused forkhead box P3 (FOXP3) protein, the "master transcriptional regulator" of regulatory T cells (Tregs), was administered into the bilateral nasal cavities of the ovalbumin (OVA)-immunoglobulin E mouse model of AR. The effects of these proteins on AR following OVA administration were investigated using histopathological, nasal symptom, flow cytometry, and cytokine dot blot analyses. Polyarginine-mediated FOXP3 protein transduction induced Treg-like cell generation in the nasal epithelium and allergen tolerance. Overall, this study proposes FOXP3 activation-mediated Treg induction as a novel and potential therapeutic strategy for AR, providing a potential alternative to conventional intranasal drug application for nasal drug delivery.

Negatively charged phospholipids doped liposome delivery system for mRNA with high transfection efficiency and low cytotoxicity

Messenger RNA (mRNA) has become one of the most potential drugs in recent years. However, efficient and safe delivery of fragile and easily degradable mRNA is a major challenge. Appropriate delivery system (DS) determines the final effect of mRNA. Cationic lipids play a crucial and decisive role in the entire DS, but also cause huge biosafety problems due to the high toxicity. In this study, a new DS for mRNA delivery that combines negatively charged phospholipids was developed in order to neutralize the positive charge and thus increase the safety. Further, the factors affecting mRNA transfection from cell to animal were investigated. The mRNA DS with optimum condition of lipid composition, proportions, structure, and transfection time was synthesized. Adding an appropriate amount of the anionic lipid to liposomes could increase the safety while maintaining the original transfection efficiency. For transporting mRNA in vivo, requirements regarding the mRNA encapsulation and releasing rate should be further considered to optimize DS design and preparation.

Protective RNA nanovaccines against Mycobacterium avium subspecies hominissuis.

The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and DOTAP (dioleoyl 3 trimethylammonium propane), hence named QTAP, for the efficient delivery of mRNA vaccine constructs into cells. Electron microscopy indicated that the complexation of mRNA with QTAP forms nanoparticles with an average size of 75 nm and which have ~90% encapsulation efficiency. The incorporation of pseudouridine-modified mRNA resulted in higher transfection efficiency and protein translation with low cytotoxicity than unmodified mRNA. When QTAP-mRNA or QTAP alone transfected macrophages, pro-inflammatory pathways (e.g., NLRP3, NF-kb, and MyD88) were upregulated, an indication of macrophage activation. In C57Bl/6 mice, QTAP nanovaccines encoding Ag85B and Hsp70 transcripts (QTAP-85B+H70) were able to elicit robust IgG antibody and IFN- ɣ, TNF-α, IL-2, and IL-17 cytokines responses. Following aerosol challenge with a clinical isolate of M. avium ss. hominissuis (M.ah), a significant reduction of mycobacterial counts was observed in lungs and spleens of only immunized animals at both 4- and 8-weeks post-challenge. As expected, reduced levels of M. ah were associated with diminished histological lesions and robust cell-mediated immunity. Interestingly, polyfunctional T-cells expressing IFN- ɣ, IL-2, and TNF- α were detected at 8 but not 4 weeks post-challenge. Overall, our analysis indicated that QTAP is a highly efficient transfection agent and could improve the immunogenicity of mRNA vaccines against pulmonary M. ah, an infection of significant public health importance, especially to the elderly and to those who are immune compromised.

The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection.

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed VHHs and the antigen present in the cell, preventing the necessity to remove the excess unbound VHHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH

Front Cover: Polypiperazines are hydrophilic pH-sensitive polymers reaching high transfection efficiency and guaranteeing good biocompatibility. They are almost charge-neutral under physiological conditions avoiding unintended side effects, while the isopropyl-modified polypiperazine outperforms the commercial linear PEI at low N/P ratios. qPCR revealed that pDNA can still be amplified while complexed with piperazine-based polymers, providing a promising platform for effective polymeric gene delivery systems. This is reported by Anja Traeger, Johannes C. Brendel, and co-workers in article number 2200517.

Cobweb‐Inspired Microenvironment‐Targeting Nanosystem with Sequential Multiple‐Stage Stimulus‐Response Capacity for Ischaemic Tissue Repair

AbstractMyocardial ischaemia is pathologically complicated; various changes in intracellular and extracellular microenvironments make it essential to develop a smart drug system with multiple stimulus responses to adapt to the complex process. Inspired by the cobweb, this study designs a microreticular nanosystem that adheres to tissue and is sequentially responsive to multiple stimuli in the ischaemic microenvironment. The nanosystem is fabricated from hyaluronic acid (HA), ROS‐responsive B‐PDEA, and hypoxia‐sensitive VEGF‐expressing plasmids (EPODNA) through electrostatic interactions. After intramyocardial injection, the tissue‐adhesive property of the nanosystem will significantly decrease its acute loss from the injection site. Extracellularly, the microreticular nanosystem first responds to activated hyaluronidase (hyal), releasing HA for microenvironment regulation and B‐PDEA/DNA nanoparticles (NP) with high transfection efficiency for cardiac cells. Intracellularly, ROS sequentially induced B‐PDEA/DNA NP dissociation, consuming some ROS to attenuate oxidative stress and releasing DNA to promote its expression. Meanwhile, local hypoxia significantly activates VEGF expression in plasmids for myocardial revascularization and repair. The function of the microreticular nanosystem is systematically evaluated in vitro. In a rat model of myocardial infarction, treatment with the microreticular nanosystem significantly promotes functional and structural improvements. Collectively, the study provides a promising smart nanosystem to promote tissue repair after complex damage.

Establishment of an Antiplasmodial Vaccine Based on PfRH5-Encoding RNA Replicons Stabilized by Cationic Liposomes.

Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.

Efficient transfected liposomes co-loaded with pNrf2 and pirfenidone improves safe delivery for enhanced pulmonary fibrosis reversion

Pulmonary fibrosis (PF) is an interstitial lung disease with complex pathological mechanism, and there is currently a lack of therapeutics that can heal it completely. Using gene therapy with drugs provides promising therapeutic strategies for synergistically reversing PF. However, improving the intracellular accumulation and transfection efficiency of therapeutic nucleic acids is still a critical issue that urgently needs to be addressed. Herein, we developed lipid nanoparticles (PEDPs) with high transfection efficiency coloaded with pDNA of nuclear factor erythroid 2-related factor 2 (pNrf2) and pirfenidone (PFD) for PF therapy. PEDPs can penetrate biological barriers, accumulate at the target, and exert therapeutic effects, eventually alleviating the oxidative stress imbalance in type II alveolar epithelial cells (AECs II) and inhibiting myofibroblast overactivation through the synergistic effects of Nrf2 combined with PFD, thus reversing PF. In addition, we systematically engineered various liposomes (LNPs), demonstrated that reducing the polyethylene glycol (PEG) proportion could significantly improve the uptake and transfection efficiency of the LNPs, and proposed a possible mechanism for this influence. This study clearly reveals that controlling the composition ratio of PEG in PEDPs can efficiently deliver therapeutics into AECs II, improve pNrf2 transfection, and synergize with PFD in a prospective strategy to reverse PF.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Iron Oxide Nanoparticle-Mediated siRNA Delivery System for Huntington’s Disease Treatment

Huntington’s disease (HD) is an autosomal dominant disease affecting neurons predominantly in the striatum due to the production of the toxic huntingtin protein. Lowering the concentration of mutant huntingtin is a promising therapeutic approach, and a suitable delivery system is fascinating. Nanoparticles (NPs) minimize the host immune response and have no limit concerning the number of NPs administered. They are safe, targeted, and effective for RNA therapeutics providing a significant mode to cross the blood–brain barrier for a broad range of clinical applications. The present study generated and characterized magnetic NPs (MNPs) using the co-precipitation method with a mean particle size of around 10–20 nm. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. These oleic acid-coated MNPs were further cross-linked with polyethyleneimine and designed to deliver interfering RNA into human embryonic kidney cells (HEK-293) driven by an external magnetic field. These MNPs showed low cytotoxicity with high transfection efficiency. Furthermore, a transient downregulation was observed in endogenous huntingtin protein obtained by RT-PCR and Western blot analysis. Thus, these MNPs may represent a promising and efficient platform for siRNA delivery and provide a potential treatment strategy for HD.

Engineering high post-electroporation viabilities and transfection efficiencies for elongated cells on suspended nanofiber networks

The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.

Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells

Although surgery-based comprehensive therapy is becoming the main approach to treat laryngeal cancer, recurrence, metastasis, radiotherapy resistance and chemotherapy tolerance are still the main causes of death in patients. Targeted inhibition of laryngeal cancer stem cells has been considered as the consensus to cure laryngeal cancer. Our previous study has confirmed proto-oncogene Bmi-1 as a key regulator for self-renewal of laryngeal cancer stem cells. Targeted knockdown of Bmi-1 gene effectively inhibited the self-renewal and differentiation of laryngeal cancer stem cells, leading to the promoted sensitivity to chemotherapy including paclitaxel. However, due to off-target effects and quick degradation of the naked Bmi-1-RNAi small RNA oligo by nuclease in body fluids, it is urgently needed to develop a tumor-targeted delivery system with a protective shell. In this study, we designed and synthesized cRGD peptide-modified chitosan-polyethylene glycol slow-release nanoparticles (mPEG-CS-cRGD/Bmi-1RNAi-PTX) containing Bmi-1RNAi siRNA oligo and paclitaxel, which showed spherical in shape, 200 nm diameter in size, low cytotoxicity, strong DNA wrapping, resistance to nuclease degradation and high transfection efficiency to cells. Functional analysis indicated significant suppression of cell proliferation and migration and induction of apoptosis by the nanocomplex in laryngeal cancer cells in vitro. By application to the mouse model with laryngeal cancer, the nanocomplex inhibited tumor growth significantly in vivo. In addition, cRGD peptide, paclitaxel and Bmi-1 siRNA in the nanoparticles showed synergistic effects to suppress laryngeal cancer stem cells. In conclusion, this study not only developed a laryngeal tumor-targeted chemotherapeutic system, but also demonstrated a Bmi-1 RNAi-based chemotherapeutic strategy to inhibit cancer stem cells, having strong potential to treat laryngeal cancer patients suffering therapy resistance and/or tumor recurrence.

Nano-carrier for gene delivery and bioimaging based on pentaetheylenehexamine modified carbon dots

Carbon dots (CDs) have attracted much attention due to their excellent properties and applications, especially the use for gene delivery. Considering the risks and concerns involved in the use of viral vectors for gene delivery in vivo, non-viral vectors such as CDs have gradually become an ideal alternative due to their biocompatibility and low toxicity. Therefore, in this study, the potential to apply CDs as a non-viral vector for gene delivery was investigated. The CDs were prepared using citric acid and pentaethylenehexamine (PEHA) as precursors via a one-step microwave-mediated approach. The optical, structural, and morphological properties of PEHA-derived CDs (PCDs) were characterized by ultra-violet spectroscopy (UV–vis), photoluminescence (PL), Fourier Transform Infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), zeta potential, circular dichroism spectrometry, atomic force (AFM) and transmission electron microscopies (TEM). The analysis demonstrated that the as-prepared PCDs were rich in amine groups and were positively charged. Subsequently, gel retardation assay showed that PCDs could non-covalently bind with DNA at a mass ratio of 2:1 (PCDs: DNA). Additionally, PCDs possessed a tremendously lower cytotoxicity compared with polyethylenimine (PEI), a popular precursor/dopant for many CDs preparations, and their plasmid composite showed a high transfection efficiency. Meanwhile, PCDs were also observed to cross the blood–brain barrier (BBB) by using a zebrafish model. In conclusion, these results significantly indicate that PCDs are a potential non-viral nucleic acid/gene vector to gene therapy. Also, PCDs can be utilized in drug delivery for treating brain diseases, such as Alzheimer’s disease and brain tumors.

Glycopolymers Mediate Suicide Gene Therapy in ASGPR-Expressing Hepatocellular Carcinoma Cells in Tandem with Docetaxel.

Cationic glycopolymers stand out as gene delivery nanosystems due to their inherent biocompatibility and high binding affinity to the asialoglycoprotein receptor (ASGPR), a target receptor overexpressed in hepatocellular carcinoma (HCC) cells. However, their synthesis procedure remains laborious and complex, with problems of solubilization and the need for protection/deprotection steps. Here, a mini-library of well-defined poly(2-aminoethyl methacrylate hydrochloride-co-poly(2-lactobionamidoethyl methacrylate) (PAMA-co-PLAMA) glycopolymers was synthesized by activators regenerated by electron transfer (ARGET) ATRP to develop an efficient gene delivery nanosystem. The glycoplexes generated had suitable physicochemical properties and showed high ASGPR specificity and high transfection efficiency. Moreover, the HSV-TK/GCV suicide gene therapy strategy, mediated by PAMA144-co-PLAMA19-based nanocarriers, resulted in high antitumor activity in 2D and 3D culture models of HCC, which was significantly enhanced by the combination with small amounts of docetaxel. Overall, our results demonstrated the potential of primary-amine polymethacrylate-containing-glycopolymers as HCC-targeted suicide gene delivery nanosystems and highlight the importance of combined strategies for HCC treatment.

Surfactant like peptides for targeted gene delivery to cancer cells

Surfactant like peptides (SLPs) are a class of amphiphilic peptides widely used for drug delivery and tissue engineering. However, there are very few reports on their application for gene delivery. The current study was aimed at development of two new SLPs, named (IA)4K and (IG)4K, for selective delivery of antisense oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) to cancer cells. The peptides were synthesized by Fmoc solid phase synthesis. Their complexation with nucleic acids was studied by gel electrophoresis and DLS. The transfection efficiency of the peptides was assessed in HCT 116 colorectal cancer cells and human dermal fibroblasts (HDFs) using high content microscopy. The cytotoxicity of the peptides was assessed by standard MTT test. The interaction of the peptides with model membranes was studied using CD spectroscopy. Both SLPs delivered siRNA and ODNs to HCT 116 colorectal cancer cells with high transfection efficiency which was comparable to the commercial lipid-based transfection reagents, but with higher selectivity for HCT 116 compared to HDFs. Moreover, both peptides exhibited very low cytotoxicity even at high concentrations and long exposure time. The current study provides more insights into the structural features of SLPs required for nucleic acid complexation and delivery and can therefore serve as a guide for the rational design of new SLPs for selective gene delivery to cancer cells to minimize the adverse effects in healthy tissues.

Self‐Fluorescent Reactive Oxygen Species‐Responsive Zinc(II)‐Quinoline Module Functionalized Polycations Hold Great Potentials in siRNA‐Mediated Gene Therapy

AbstractA novel quinoline‐based thioketal‐containing zinc (II) coordinative module (Zn‐QS) is designed to transform common low‐molecular‐weight (LMW) polyethyleneimine (PEI) into a high‐performance siRNA carrier (Zn‐PQ) with self‐fluorescence and reactive oxygen species (ROS)‐responsiveness. In the multifunctional module, a quinoline derivative, frequently serving as pharmacophore, is utilized as the highly biocompatible ligand, and the Zn (II) coordinative quinoline‐based ligand also provides the robust self‐fluorescence character. And the ROS‐responsive thioketal spacer is introduced to trigger the siRNA controlled release into cytoplasm. Consequently, by the synergistic effects between Zn‐QS and LMW PEI on improving several crucial transfection‐efficiency‐related steps, the representative Zn‐PQ4 exhibits very high transfection efficiency of 1.8 and 2.5‐folds commercial Lipo2k and PEI25k in stem cell rADSC, respectively, >90% of transfection rates in a variety of conventional cell lines (3T3, HK‐2, HepG2, 293T, HeLa, PANC‐1), and 98.4% and 99.9% EGFP knockdown rates in 293T and HepG2 cells, respectively. Furthermore, Zn‐PQ4/siRNA polyplexes can circumvent various obstacles existing in systemic application to accumulate in heart, liver, and kidney and greatly silence the DNA expression in vivo. This work demonstrates the promising application prospects of the resulting efficient siRNA carriers in siRNA‐mediated gene therapy and fluorescence‐guided research.

Anticancer effect of miR-124-loaded liposomal nanoparticles on pancreatic cancer progression

Pancreatic cancer (PC) is a common malignancy that is characterized by strong invasiveness and rapid development. With lack of effective treatments, patients still suffer from poor outcome. In this study, we established liposomal nanoparticles carrying microRNA-124 (Ls/miR-124) and detected their inhibitory effect on PC cells. Ls/miR-124 nanoparticles were prepared and co-cultured with PC cells (Ls/miR-124 group), as some PC cells were treated with pure liposome particles (Ls group), or gemcitabine (gemcitabine group) and some untreated were treated as control group. Flow cytometry and transwell assay were used to determine apoptosis and migration of PC cells. Bax, Bcl-2, and Caspase-3 expressions were measured. The Ls/miR-124 nanoparticles presented around 100 nm size at 3:1 molar ratio of DOTAP/Chol, with stable properties and high potential. The fluorescence intensity of the cells in the Ls/miR-124 group and gemcitabine group were higher than Ls and blank groups. RT-qPCR analysis confirmed the up-regulation of miR-124 with high transfection efficiency of 85%. Proliferation of cells in each group increased upon culture. Administration of either Ls/miR-124 nanoparticles or gemcitabine significantly decreased PC cell proliferation and increased apoptosis at similar apoptotic rate of 29.76±3.78% and 30.15±3.57. Besides, the Ls/miR-124 and gemcitabine groups had cell cycle arrest in G0/G1 phase (67.98±3.45%) and exhibited decreased migration of 19.78±2.97% and invaded cells (42.56±6.98%). The expression of Bax in both groups decreased, while expressions of Bcl-2 and Caspase-3 increased. In conclusion, administration of Ls/miR-124 nanoparticles decreased cell viability and migration of PC cells, inhibiting cell cycle progression but increasing apoptosis and restraining development of PC.

PEGylated reduced graphene oxide as nanoplatform for targeted gene and drug delivery

Combination therapies involving anticancer drugs and gene therapy agents have become a potential treatment for different cancer types reducing the side effects of anticancer drugs while enhancing their effectiveness. Here, we improved our previously published strategy that uses multiple pyrene groups of poly(ethylene glycol) (PEG) brushed graft copolymer to enhance the biological use of reduced graphene oxide (rGO) and introduced an azide containing monomer in the copolymer for further modifications to carry hydrophobic drugs like doxorubicin (Dox) along with oligonucleotides such as small interfering RNAs (siRNA). Azide functionality on the side chain of coated copolymer enables not only the conjugation of alkyne end group functional cationic copolymer but also allows the addition of targeting moieties like EPPT1 peptide that selectively binds MUC1, an early hallmark of tumorigenesis. To achieve the outlined carrier, first, a well-defined PEG, pyrene, azide, and methyl methacrylate containing poly[(poly(ethylene glycol) methyl ether methacrylate)-co-(3-azidopropyl methacrylate)-co-(methyl methacrylate)-co-(1-pyrenemethyl methacrylate)] (P(PEGMA-co-AzPMA-co-MMA-co-PMA), PAMP) copolymer was synthesized via ATRP and through its azide group another well-defined, cationic copolymer composed of hexyl methacrylate, 2-dimethyl amino ethyl methacrylate, and N,N,N-trimethylaminoethyl methacrylate iodide (P(HMA-co-DMAEMA-co-TMAEMA) was coupled via copper-catalyzed azide-alkyne cycloaddition (CuAAC). This cationic copolymer was synthesized using a unique ATRP initiator to allow click reaction as described as well as pH-dependent degradation through its hydrazone linkage. The obtained graft copolymer (PAMP-CP) was then coated on a graphene oxide (GO) surface and GO was in situ reduced to rGO (PAMP-CP-rGO) to have an rGO-based PEGylated nanoplatform that can be highly loaded with doxorubicin and successfully condensed with anti-GAPDH siRNA. Consequently, this dual drug delivery nanoplatform showed a controlled drug release that switches between release kinetics by the change of pH and high transfection efficiency regarding targeted GAPDH protein levels. Moreover, in vitro cytotoxicity tests showed that EPPT1-targeted Dox-loaded PAMP-CP-rGO shows concentration-dependent and selective cellular toxicity against cancer cells compared to its non-targeted versions. These results demonstrated the potential of a highly adaptable, multifunctional nanoplatform for safely and selectively transporting different cargoes to the tumor site.