Delivery Of Therapeutic siRNA Research Articles

Design of Experiments Grants Mechanistic Insights into the Synthesis of Spermine-Containing PBAE Copolymers.

Successful therapeutic delivery of siRNA with polymeric nanoparticles seems to be a promising but not vastly understood and complicated goal to achieve. Despite years of research, no polymer-based delivery system has been approved for clinical use. Polymers, as a delivery system, exhibit considerable complexity and variability, making their consistent production a challenging endeavor. However, a better understanding of the polymerization process of polymer excipients may improve the reproducibility and material quality for more efficient use in drug products. Here, we present a combination of Design of Experiment and Python-scripted data science to establish a prediction model, from which important parameters can be extracted that influence the synthesis results of polybeta-amino esters (PBAEs), a common type of polymer used preclinically for nucleic acid delivery. We synthesized a library of 27 polymers, each one at different temperatures with different reaction times and educt ratios using an orthogonal central composite (CCO-) design. This design allowed a detailed characterization of factor importance and interactions using a very limited number of experiments. We characterized the polymers by analyzing the resulting composition by 1H-NMR and the size distribution by GPC measurements. To further understand the complex mechanism of block polymerization in a one-pot synthesis, we developed a Python script that helps us to understand possible step-growth steps. We successfully developed and validated a predictive response surface and gathered a deeper understanding of the synthesis of polyspermine-based amphiphilic PBAEs.

Evaluation of lipopolymer as therapeutic siRNA delivery agents in non-malignant and malignant primary cells.

e15189 Background: Targeting leukemia-driving oncogenes via short interfering RNA (siRNA) offers an alternative approach to leukemia treatment that can overcome the limitations of current therapies. Low molecular weight polyethyleneimine grafted with lipids (lipopolymers) have emerged as promising carriers for siRNA delivery to leukemia cells. Lipopolymers harboring select lipids were recently shown to downregulate target oncogene leading to significant anti-leukemic activity in both in vitro and in vivo cell line-based leukemia models. The goal of this study was to evaluate these lipopolymers in (i) PBMCs obtained from healthy donors to determine the potential impact on non-malignant cells, and (ii) primary leukemia patient cells to assess therapeutic efficacy. Methods: Target gene levels were determined by RT-qPCR and the effect of treatment on progenitor cells was assessed by measuring changes in colony forming ability. Results: At day 1 post-treatment, no changes in FLT3 mRNA levels were observed in all 6 PBMC samples with the 2 candidate lipopolymers (LeuFectB and LeuFectC) tested. Among 11 PBMC samples evaluated at day 2, only one sample showed a significant reduction in oncogene transcript levels on treatment with LeuFectB. The impact of lipopolymer/siRNA complex treatment on progenitor cells in the PBMC population was then assessed. Of the 7 PBMC samples tested, significant reduction in colony numbers was observed in one sample with LeuFectB and two samples with LeuFectC. On an average, the lipopolymer/siRNA complexes were well tolerated by normal healthy cells as changes observed in oncogene expression and colony forming ability were not consistent across the whole pool. Primary acute lymphoblastic leukemia (ALL) patient cells treated with complexes targeting STAT5A showed marked reduction in STAT5A transcript levels in 4 out of 7 samples with both the lipopolymers tested. The subsequent impact of STAT5A downregulation on the clonogenicity of leukemia stem cells was observed in the significant decrease in colony numbers seen in 3 samples (out of 8) with LeuFects. Additionally, combined downregulation of STAT5A and BCR-ABL genes in BCR-ABL+ ALL patient samples led to a notable decrease in cell proliferation and viability. Moreover,2 out of 3 acute myeloid leukemia (AML) patient samples harboring the FLT3-ITD mutation showed significant retardation in their colony forming ability on treatment with FLT3-targeting siRNA complexes. Collectively, the ALL and AML primary samples demonstrated promising preliminary results, suggesting a therapeutic effect on the stem cell population which is critical for mitigating relapse or prolonging remission. Conclusions: These findings provide further proof-of-concept for clinical translation of lipopolymer mediated siRNA therapy of leukemia, showing relative safety and selectivity of these lipopolymer/siRNA complexes.

Effects of Histidine Oligomers in Lipid Nanoparticles on siRNA Delivery.

In this study, we developed histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) was greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of approximately 65nm demonstrated a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibited a significant three-fold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintained comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibited excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs. This article is protected by copyright. All rights reserved.

Abstract 650: Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer

Abstract Triple-negative Breast cancer (TNBC) is characterized by the absence of ER, PR, and HER-2 receptors and is associated with poor prognosis, early relapse, and distant metastasis. In neoadjuvant setting, most patients (~50-70%) with TNBC do not achieve complete remission. Currently, there is no effective targeted therapy for TNBC patients. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. PIM3 is a serine/threonine kinase and is upregulated in leukemia and various solid cancers. Although PIM1 has been shown to play a critical role in cell proliferation in cMyc-enriched TNBC tumors, the role of PIM3 in TNBC is not known. To determine the clinical significance of PIM3 in TNBC patient prognosis and progression, we performed a Kaplan-Meier analysis in the TCGA TNBC patient database. We found that PIM3 expression is significantly associated with shorter patient survival in TNBC patients (n=77, p=0.0088). PIM3 protein is highly expressed in TNBC cells compared to normal breast epithelium and other ER+ and HER2+ breast cancer subtypes. Inhibition of PIM3 by siRNA significantly suppressed TNBC cell proliferation, migration, and invasion of TNBC cells including MDA-MB-231, MDA-MB-436, and BT20 cells. Lenti-based PIM3 overexpression induced TNBC cell proliferation, invasion, and migration. Non-biased reverse phase protein array (RPPA) assay and Western blot analysis showed that PIM3 inhibition markedly reduced the expression of EF2 Kinase protein, and we found it to be associated with shorter patient survival and poor prognosis. Immunoprecipitation of PIM3 indicated that it forms a heterodimer with EF2K whose inhibition by siRNA also suppressed TNBC cell proliferation, migration, invasion, and tumor growth. Furthermore, PIM3 inhibition by siRNA significantly enhanced the antiproliferative effect of Doxorubicin, which is one of the first-line chemotherapeutics used in patients. Moreover, in vivo therapeutic delivery of PIM3 siRNA loaded albumin nanoparticles (ALNPs-PIM3 siRNA) markedly delayed tumor growth of two different TNBC tumor xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Analysis of tumors by IHC after 4 weeks of treatment demonstrated that ALNP-PIM3 siRNA treatment led to inhibition of intra-tumoral proliferation and induction of apoptosis. Currently, we are conducting an in vivo study in TNBC tumor models by combining ALNP-PIM3 siRNA therapy with doxorubicin. Overall, our studies suggest that PIM3 is a marker for poor patient survival that drives TNBC tumor growth and progression and a novel potential molecular target in TNBC. Citation Format: Pinar Atalay, Goknur Kara, Rumeysa Ozyurt, Nermin Kahraman, Bulent Ozpolat. Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 650.

Targeted siRNA delivery to lung epithelia reduces airway inflammation in a mouse model of allergic asthma

Asthma is a chronic inflammatory disease triggered by allergic reactions in the bronchia. These reactions lead to swelling of mucous membranes, hypersecretion of mucus, and bronchoconstriction, resulting in a restricted opening of the lung airway. Allergic pulmonary inflammation and airway hyperresponsiveness are induced when Th2 cytokines, such as interleukin (IL)-4 and IL-13, bind to their cognate receptors on lung epithelial cells. Specifically, IL-13 stimulates inflammation through a multi-subunit receptor, mainly the alpha chain of the IL-4 receptor (IL-4Rα), which also plays a role in IL-4 signaling. In this study, we employed a lung epithelial cell-targeting siRNA carrier composed of a rabies virus glycoprotein-derived small peptide coupled with cationic nona-arginine and trileucine before cysteine peptide (RVG9R3LC). This carrier was complexed with siRNA, enabling targeted delivery of therapeutic siRNA to IL-4Rα (siIL4Rα) expressed in lung epithelial cells within an asthma model in vivo. Our approach demonstrated efficient gene knockdown in cultured lung epithelial cells and in vivo. Furthermore, two administrations of therapeutic siIL4Rα protected the ovalbumin-sensitized and challenged asthma mouse model from airway inflammation and excessive mucus secretion. Our findings suggest that the peptide-siRNA carrier system presents a promising therapeutic approach for respiratory inflammation.Graphical abstract

Therapeutic siRNAs: small molecules with bigger function

In the realm of molecular medicine, therapeutic small-interfering RNA (siRNA) is a promising class of molecules with exciting new opportunities in healthcare. An essential component of RNA interference (RNAi) is siRNA. In clinical setup, siRNA may be engineered to specifically target genes linked to certain diseases, providing a focused and accurate method of modulating gene expression. Many important factors characterize the use of therapeutic siRNA. Target gene expression is selectively silenced by therapeutic siRNA through the use of the RNAi pathway. Guided by corresponding messenger RNA (mRNA) sequences, the double-stranded siRNA molecule is integrated into the RNA-induced silencing complex (RISC). This mechanism causes translational repression or mRNA cleavage, which in turn causes the relevant protein to be downregulated. Therapeutic siRNA is a promising avenue in several areas. siRNA can be used in oncology to target oncogenes or other important regulators, inhibiting cancer cell proliferation. Furthermore, by modifying the expression of genes linked to disease, siRNA may be used to treat viral infections, neurological conditions, and other rare and hereditary diseases. The selectivity of therapeutic siRNA is one of its main advantages. siRNA sequences can be engineered and customized to target specific genes linked to different diseases like cancer or genetic disorders. This focused strategy improves the accuracy of the therapeutic intervention and reduces off-target effects. In a recent article by Nissen et al., siRNA targeting LP(a) in a trial with 48 participants without cardiovascular disease and with lipoprotein(a) concentrations of 75 nmol/L or greater (or ≥30 mg/dL), was used to evaluate its tolerability and efficacy to reduce LP(a). Results showed that Lepodisiran was found to be well tolerated and resulted in dose-dependent, long-lasting reduction in serum lipoprotein(a) concentrations in phase 1 trial with elevated lipoprotein(a) levels. Therapeutic siRNA-based diagnosis is rapidly advancing, and several siRNA-based medications are now entering clinical trials. These trials aim to assess the pharmacokinetics, safety, and effectiveness of siRNA therapeutics in human subjects, offering crucial new information about their potential as a treatment option. Despite the therapeutic potential of siRNA, clinical translation of siRNA faces a challenge in its effective delivery. Naked siRNA molecules are more likely to degrade with a poor half-life and poor cellular uptake. Therefore, to increase the stability and delivery of therapeutic siRNA to target cells, a variety of delivery systems, such as lipid nanoparticles, viral vectors, and polymer-based carriers, are being investigated.
 

SiRNA Delivery from Cationic Nanocarriers Prepared by Diffusion-assisted Loading in the Presence and Absence of Electrostatic Interactions.

In this study, we use modified cationic nanocarriers as vehicles for the intracellular delivery of therapeutic siRNA. After developing nanocarrier formulations with appropriate pKa, size, swellability, and cytocompatibility, we investigated the importance of siRNA loading methods by studying the impact of the pH and time over which siRNA is loaded into the nanocarriers. We concentrate on diffusion-based loading in the presence and absence of electrostatic interactions. siRNA release kinetics were studied using samples prepared from nanocarriers loaded by both mechanisms. In addition, siRNA delivery was evaluated for two formulations. While previous studies were conducted with samples prepared by siRNA loading at low pH values, this research provides evidence that loading conditions of siRNA affect the release behavior. This study concludes that this concept could prove advantageous for eliciting prolonged intracellular release of nucleic acids and negatively charged molecules, effectively decreasing dose frequency and contributing to more effective therapies and improved patient outcomes. In addition, our findings could be leveraged for enhanced control over siRNA release kinetics, providing novel methods for the continued optimization of cationic nanoparticles in a wide array of RNA interference-based applications.

Restoration of Mir-185 in Combination with BCR-ABL Downregulation By Therapeutic Delivery of siRNA with Lipid Nanoparticle Carriers Targets Drug Resistant Leukemic Stem/Progenitor Cells

Background: Despite development of effective tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), TKI therapy is not curative as some patients develop drug resistance or are at risk of disease relapse. Importantly, CML leukemic stem cells (LSCs) remain largely unresponsive and represent a key population for sustaining disease and relapse. We previously demonstrated that miR-185 levels were significantly reduced in CD34 + stem/progenitor cells from TKI-nonresponders compared to TKI responders. Further, restoration of miR-185 levels in CML LSCs and their progenitors impaired their survival and sensitized them to TKI in vitro and in vivo. However, the therapeutic potential of downregulating BCR-ABL using siRNA in combination with miR-185 mimic treatment to target CML LSCs have yet to be elucidated. Objective: Downregulation of BCR-ABL using siRNA represents a promising therapeutic approach to inhibit its kinase and non-kinase functions to combat drug-resistant CML and overcome limitations of current therapy. Additionally, previous work suggests that restoring miR-185 levels may improve therapeutic outcomes from complimentary therapies in CML, particularly in combination with BCR-ABL inhibition. Therefore, we aim to investigate the therapeutic outcomes and mechanism of action of inhibiting BCR-ABL using siRNA in combination with miR-185 mimic treatment in drug-resistant CML. Methods: To assess the effects of BCR-ABL downregulation in combination with miR-185 restoration, we encapsulated siRNA targeting BCR-ABL and a miR-185 mimic using an optimized lipid nanoparticle (LNP) formulation. Biological effects and mechanistic changes of this novel RNA combination was evaluated using various CML cell lines expressing b2a2, b3a2, T315I mutated BCR-ABL and CD34 + stem/progenitor cells from non-responder patients. Results: A total of 7 siRNA sequences targeting various fusion regions of BCR-ABL were designed and tested to effectively target primitive CML cells. The highest performing BCR-ABL siRNA encapsulated showed significant knockdown in various CML models expressing b2a2, b3a2 and T315I mutated BCR-ABL (65-99% knockdown). After 48 hours of 0.5 μg/mL BCR-ABL siRNA LNP treatment, BCR-ABL protein levels and its direct downstream target p-CrkL are undetectable by immunoblotting. The robust downregulation of BCR-ABL results in a dose-dependent decrease in cell viability (down to 28% and 9.4% viable cells at a dose of 1 μg/mL) and increase in apoptotic cells (70% and 78% Annexin-V positive) in imatinib resistant K562 and T315I mutated cells, respectively after 72 hours. Similarly, encapsulated miR-185 mimic downregulates its main downstream target PAK6 transcript levels down to 50% in drug sensitive and drug-resistant cell lines and in TKI nonresponder CD34 + stem/progenitor cells. More importantly, our RNA LNP formulation demonstrated significant uptake (> 80% Cy5 positive) of fluorescent-labelled siRNA in TKI nonresponder CD34 + stem/progenitor cells and decreased BCR-ABL mRNA transcript levels by 60%. The combined BCR-ABL inhibition and miR-185 mimic treatment were determined to be highly synergistic when analysed using the Chou-Talalay (combination index less than 1), ZIP, Bliss and HSA methods (synergy scores larger than 10) in both drug-sensitive and drug-resistant CML cells. Furthermore, treatment of BCR-ABL siRNA and miR-185 using LNPs decreased the colony forming units of nonresponder patient cells by 50%. Interestingly, this effect was further enhanced when combined with TKIs, decreasing colony forming units to 20% compared to control after 14 days. Conclusion: We have uncovered a LNP formulation that is able to effectively deliver therapeutic BCR-ABL siRNA and miR-185 mimic into primitive CML cells. This combination treatment significantly reduces the levels of oncogenic BCR-ABL and PAK6, decreasing the viability clonogenic potential and increases apoptosis in CML cell line models and primary patient cells. Importantly, this combination strategy overcomes TKI resistance in CD34 + stem/progenitor cells suggesting a feasible and effective model for patients suffering from clinical resistance and relapse.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

Spermine-Based Poly(β-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.

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.

Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection.

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) invivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) lung infection invivo. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity invivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The study presented here demonstrates an attractive alternate delivery strategy for the prophylactic treatment of both future and emerging respiratory viral diseases.

Cancer Therapeutic siRNA Delivery and Imaging by Nitrogen- and Neodymium-Doped Graphene Quantum Dots.

While small interfering RNA (siRNA) technology has become a powerful tool that can enable cancer-specific gene therapy, its translation to the clinic is still hampered by the inability of the genes alone to cell transfection, poor siRNA stability in blood, and the lack of delivery tracking capabilities. Recently, graphene quantum dots (GQDs) have emerged as a novel platform allowing targeted drug delivery and fluorescence image tracking in visible and near-infrared regions. These capabilities can aid in overcoming primary obstacles to siRNA therapeutics. Here, for the first time, we utilize biocompatible nitrogen- and neodymium-doped graphene quantum dots (NGQDs and Nd-NGQDs, respectively) for the delivery of Kirsten rat sarcoma virus (KRAS) and epidermal growth factor receptor (EGFR) siRNA effective against a variety of cancer types. GQDs loaded with siRNA noncovalently facilitate successful siRNA transfection into HeLa cells, confirmed by confocal fluorescence microscopy at biocompatible GQD concentrations of 375 μg/mL. While the GQD platform provides visible fluorescence tracking, Nd doping enables deeper-tissue near-infrared fluorescence imaging suitable for both in vitro and in vivo applications. The therapeutic efficacy of the GQD/siRNA complex is verified by successful protein knockdown in HeLa cells at nanomolar siEGFR and siKRAS concentrations. A range of GQD/siRNA loading ratios and payloads are tested to ultimately provide substantial inhibition of protein expression down to 31-45%, comparable with conventional Lipofectamine-mediated delivery. This demonstrates the promising potential of GQDs for the nontoxic delivery of siRNA and genes in general, complemented by multiwavelength image tracking.

Emerging Roles of Mesenchymal Stem/Stromal-Cell-Derived Extracellular Vesicles in Cancer Therapy.

Despite the tremendous efforts of many researchers and clinicians, cancer remains the second leading cause of mortality worldwide. Mesenchymal stem/stromal cells (MSCs) are multipotent cells residing in numerous human tissues and presenting unique biological properties, such as low immunogenicity, powerful immunomodulatory and immunosuppressive capabilities, and, in particular, homing abilities. Therapeutic functions of MSCs are mediated mostly by the paracrine effect of released functional molecules and other variable components, and among them the MSC-derived extracellular vesicles (MSC-EVs) seem to be one of the central mediators of the therapeutic functions of MSCs. MSC-EVs are membrane structures secreted by the MSCs, rich in specific proteins, lipids, and nucleic acids. Amongst these, microRNAs have achieved the most attention currently. Unmodified MSC-EVs can promote or inhibit tumor growth, while modified MSC-EVs are involved in the suppression of cancer progression via the delivery of therapeutic molecules, including miRNAs, specific siRNAs, or suicide RNAs, as well as chemotherapeutic drugs. Here, we present an overview of the characteristics of the MSCs-EVs and describe the current methods for their isolation and analysis, the content of their cargo, and modalities for the modification of MSC-EVs in order for them to be used as drug delivery vehicles. Finally, we describe different roles of MSC-EVs in the tumor microenvironment and summarize current advances of MCS-EVs in cancer research and therapy. MSC-EVs are expected to be a novel and promising cell-free therapeutic drug delivery vehicle for the treatment of cancer.

Effect of tocopherol conjugation on polycation-mediated siRNA delivery to orthotopic pancreatic tumors

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with a five-year survival rate of around 10 %. CXCR4 and STAT3 display crucial effects on proliferation, metastasis, angiogenesis, and formation of immunosuppressive microenvironment in pancreatic tumors. Here, we have tested the hypothesis that conjugation of α-tocopherol (TOC) to a polycation (PAMD), synthesized from CXCR4-antagonist AMD3100, will improve delivery of therapeutic siRNA to silence STAT3 in PDAC tumors. PAMD-TOC/siSTAT3 nanoparticles showed superior anti-cancer and anti-migration performance compared to the parent PAMD/siSTAT3 nanoparticles in both murine and human PDAC cell lines. The biodistribution of the nanoparticles in orthotropic mouse KPC8060 and human PANC-1 models, indicated that tumor accumulation of PAMD-TOC/siRNA nanoparticles was improved greatly as compared to PAMD/siRNA nanoparticles. This improved cellular uptake, penetration, and tumor accumulation of PAMD-TOC/siSTAT3 nanoparticles, also contributed to the suppression of tumor growth, metastasis and improved survival. Overall, this study presents a prospective treatment strategy for PDAC.

Recent Advancements in the Design of Nanodelivery Systems of siRNA for Cancer Therapy.

RNA interference (RNAi) has increased the possibility of restoring RNA drug targets for cancer treatment. Small interfering RNA (siRNA) is a promising therapeutic RNAi tool that targets the defective gene by inhibiting its mRNA expression and stopping its translation. However, siRNAs have flaws like poor intracellular trafficking, RNase degradation, rapid kidney filtration, off-targeting, and toxicity, which limit their therapeutic efficiency. Nanocarriers (NCs) have been designed to overcome such flaws and increase antitumor activity. Combining siRNA and anticancer drugs can give synergistic effects in cancer cells, making them a significant gene-modification tool in cancer therapy. Our discussion of NCs-mediated siRNA delivery in this review includes their mechanism, limitations, and advantages in comparison with naked siRNA delivery. We will also discuss organic NCs (polymers and lipids) and inorganic NCs (quantum dots, carbon nanotubes, and gold) that have been reported for extensive delivery of therapeutic siRNA to tumor sites. Finally, we will conclude by discussing the studies based on organic and inorganic NCs-mediated siRNA drug delivery systems conducted in the years 2020 and 2021.

Insights on Development Aspects of Polymeric Nanocarriers: The Translation from Bench to Clinic.

Scientists are focusing immense attention on polymeric nanocarriers as a prominent delivery vehicle for several biomedical applications including diagnosis of diseases, delivery of therapeutic agents, peptides, proteins, genes, siRNA, and vaccines due to their exciting physicochemical characteristics which circumvent degradation of unstable drugs, reduce toxic side effects through controlled release, and improve bioavailability. Polymers-based nanocarriers offer numerous benefits for in vivo drug delivery such as biocompatibility, biodegradability, non-immunogenicity, active drug targeting via surface modification, and controlled release due to their pH—and thermosensitive characteristics. Despite their potential for medicinal use, regulatory approval has been achieved for just a few. In this review, we discuss the historical development of polymers starting from their initial design to their evolution as nanocarriers for therapeutic delivery of drugs, peptides, and genes. The review article also expresses the applications of polymeric nanocarriers in the pharmaceutical and medical industry with a special emphasis on oral, ocular, parenteral, and topical application of drugs, peptides, and genes over the last two decades. The review further examines the practical, regulatory, and clinical considerations of the polymeric nanocarriers, their safety issues, and directinos for future research.

Nanoparticles-Based Strategies to Improve the Delivery of Therapeutic Small Interfering RNA in Precision Oncology.

Small interfering RNA (siRNA) can selectively suppress the expression of disease-causing genes, holding great promise in the treatment of human diseases, including malignant cancers. In recent years, with the development of chemical modification and delivery technology, several siRNA-based therapeutic drugs have been approved for the treatment of non-cancerous liver diseases. Nevertheless, the clinical development of siRNA-based cancer therapeutics remains a major translational challenge. The main obstacles of siRNA therapeutics in oncology include both extracellular and intracellular barriers, such as instability under physiological conditions, insufficient tumor targeting and permeability (particularly for extrahepatic tumors), off-target effects, poor cellular uptake, and inefficient endosomal escape. The development of clinically suitable and effective siRNA delivery systems is expected to overcome these challenges. Herein, we mainly discuss recent strategies to improve the delivery and efficacy of therapeutic siRNA in cancer, including the application of non-viral nanoparticle-based carriers, the selection of target genes for therapeutic silencing, and the combination with other therapeutic modalities. In addition, we also provide an outlook on the ongoing challenges and possible future developments of siRNA-based cancer therapeutics during clinical translation.

Delivery of therapeutic small interfering RNA: The current patent-based landscape

Implementing small interfering RNA (siRNA) is a promising therapy because it silences disease-related genes theoretically. However, the efficient delivery of siRNA is challenging, which limits its therapeutic applications. Various pharmaceutical delivery systems containing key technologies have been developed and patented, which are of great concern to developers in the field. Despite numerous studies devoted to siRNA-delivery technologies, few researchers have systematically examined relevant patents. Patents, as bridges connecting academic progress with applicable innovation, encapsulate cumulative technological innovations and provide valuable information for academic research and commercial development. This study aims to analyze advances in therapeutic siRNA delivery technology from a patent perspective. A total of 11,509 patent documents from 3,309 patent families were collected, classified into 10 technological categories, and comprehensively analyzed. An overall patent landscape of siRNA delivery was presented from the temporal, spatial, organizational, and technological dimensions. This work is expected to help researchers and developers in the field of siRNA delivery form a basis for decision-making by combining our findings with supplementary data.

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX—the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1208/s12248-021-00653-2.