Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems.

Small interfering RNAs (siRNAs) have emerged as a powerful tool to manipulate gene expression in vitro. However, their potential therapeutic application encounters significant challenges, such as degradation in vivo, limited cellular uptake, and restricted biodistribution, among others. This study evaluates the siRNA delivery efficiency of three different lipid-substituted polyethylenimine (PEI)-based carriers, named Leu-Fect A-C, to different organs in vivo, including xenograft tumors, when injected into the bloodstream of mice. The siRNA analysis was undertaken by stem-loop RT-PCR, followed by qPCR or digital droplet PCR. Formulating siRNAs with a Leu-Fect series of carriers generated nanoparticles that effectively delivered the siRNAs into K652 and MV4-11 cells, both models of leukemia. The Leu-Fect carriers were able to successfully deliver BCR-Abl and FLT3 siRNAs into leukemia xenograft tumors in mice. All three carriers demonstrated significantly enhanced siRNA delivery into organs other than the liver, including the xenograft tumors. Preferential biodistribution of siRNAs was observed in the lungs and spleen. Among the delivery systems, Leu-Fect A exhibited the highest biodistribution into organs. In conclusion, lipid-substituted PEI-based delivery systems offer improvements in addressing pharmacokinetic challenges associated with siRNA-based therapies, thus opening avenues for their potential translation into clinical practice.

Improved delivery of Mcl-1 and survivin siRNA combination in breast cancer cells with additive siRNA complexes.

This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.

Lipopolymer/siRNA complexes engineered for optimal molecular and functional response with chemotherapy in FLT3-mutated acute myeloid leukemia.

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. STATEMENT OF SIGNIFICANCE: We show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the 'gold-standard' delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated.

Lipopolymer mediated siRNA delivery targeting aberrant oncogenes for effective therapy of myeloid leukemia in preclinical animal models.

The clinical development of tyrosine kinase inhibitors (TKI) has led to great strides in improving the survival of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. But even the new generation TKIs are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative therapeutic approaches. In contrast to exploiting proteins as targets like most conventional drugs and TKIs, RNA Interference (RNAi) exerts its therapeutic action towards disease-driving aberrant genes. To realize the potential of RNAi, the major challenge is to efficiently deliver the therapeutic mediator of RNAi, small interfering RNA (siRNA) molecules. In this study, we explored the feasibility of using aliphatic lipid (linoleic acid and lauric acid)-grafted polymers (lipopolymers) for the delivery of siRNAs against the FLT3 oncogene in AML and BCR-ABL oncogene in CML. The lipopolymer delivered siRNA potently suppressed the proliferation AML and CML cells via silencing of the targeted oncogenes. In both AML and CML subcutaneous xenografts generated in NCG mice, intravenously administered lipopolymer/siRNA complexes displayed significant inhibitory effect on tumor growth. Combining siFLT3 complexes with gilteritinib allowed for reduction of effective drug dosage, longer duration of remission, and enhanced survival after relapse, compared to gilteritinib monotherapy. Anti-leukemic activity of siBCR-ABL complexes was similar in wild-type and TKI-resistant cells, and therapeutic efficacy was confirmed in vivo through prolonged survival of the NCG hosts systemically implanted with TKI-resistant cells. These results demonstrate the preclinical efficacy of lipopolymer facilitated siRNA delivery, providing a novel therapeutic platform for myeloid leukemias.

Effect of soft and prolonged Graeco-Arabic massage in low back pain - A randomized controlled clinical trial.

BACKGROUND: Low back pain (waja-uz-zahr) due to lumbar spondylosis affects approximately 60-80% of the population at any point of life. The present study was aimed to evaluate the efficacy of soft and prolonged massage with roghan-i-suranjan on low back pain. MATERIAL AND METHODS: Clinically and radiologically diagnosed patients of low back pain were randomized into test and control groups consisting 20 patients in each group. The patients in test group received soft and prolonged massage with roghan-i-suranjan; while, in control group, short wave diathermy was given on every alternate day up to 20 min for 3 weeks. The patients were assessed before and after the treatment using the VAS and ODI (OLBP) scales. RESULTS: VAS score was reduced to 42.14%, (p < 0.001) in massage and 13.94%, (p < 0.008) in control group after the treatment [Percentual Change (PC) between the groups = 54.35%, p < 0.001]. OLBP score was improved to 37.16% (p < 0.001) in massage and 5.93% (p < 0.0012) in control group after the treatment [PC between the groups = 29.57%, p < 0.02]. CONCLUSION: Apart from the muhallil-i-awram (anti-inflammatory), mulattif (attenuative), daf-i-alam (analgesic), and musakkin (sedative) activities of the tested-drugs, the specific strokes soft and prolonged massage further enhances the evacuative and rejuvenative process through its own action of tahleel (dissolution), talteef (attenuation), taskeen (sedation), and taskheen (diaphoresis) induced by the various manipulations used in this massage. Soft and prolonged massage with tested oil was found effective in reducing the pain and improving the quality of life in the patients of low back pain.

Multiple gene knockdown strategies for investigating the properties of human leukemia stem cells and exploring new therapies.

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients, many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus, overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example, with rare, propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore, it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms, and their key pathways, so that CML LSCs can be selectively targeted or sensitized to TKIs. Here, we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI), which can be further explored as therapeutic modalities against LSCs in the context of human leukemia.

Effect of Arjun Chal on cardiovascular risk factors - a randomized controlled clinical trial.

OBJECTIVES: Cardiovascular diseases have a multifaceted, it causes modern epidemic; Recognizing in the risk factor stage, is crucial, given the risk of progression to cardiovascular disease. Ibn Sina, described CVDs as a resultant of gradual derangement of Quwwat ghadhiya (Nutritive faculty); in which management with ghidha' (diet), tadabir (regimens), dawa' (drug) has been received. To evaluate the effect of Arjun Chal (Terminalia arjuna) in CVD risk factors. And to evaluate the drug safety. METHODS: This is a randomized controlled clinical trial. Total 120 patients were screened at OPD of NIUM hospital, Bangalore during 2018-19, only 48 patients fulfilled the inclusion criteria and signed written informed consent and their detailed medical history was recorded. Arjun Chal powder (5 gm BD) for eight weeks administered in test group (n=24), Amlodipine (5 mg) and Atorvastatin (10 mg) once a day for same duration administered in control group (n=24). Efficacy of the drug assessed by the Lipid profile, BP and BMI; lipid profile were performed at baseline and at 8 weeks, while BP and BMI performed at baseline, 15, 30, 45, and 60th day intervals. Study was completed by 40 patients. The results of both the therapies were then compared and statistically analyzed. RESULTS: Totally, both groups reduces assessment parameters i.e. statistically highly significant (p<0.001). Test group showed greater reduction in terms of all assessment parmeters. But, the difference between both the groups was statistically non-significant p>0.05. CONCLUSIONS: Both test and control drugs were effective, but Arjun Chal had a slight edge over amlodipne and atorvastatin, and was found to be safe and well tolerated. It has a cardio protective potential and hence effective to delay/prevent CVD in patient with cardiovascular risk factor.Keywords: Unani System of Medicine; T. arjuna; Arjun Chal; Efficacy; Safety; Cardiovascular risk factor.

Investigation of water-insoluble hydrophobic polyethylenimines as RNAi vehicles in chronic myeloid leukemia therapy.

The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.

At the Intersection of Biomaterials and Gene Therapy: Progress in Non-viral Delivery of Nucleic Acids.

Biomaterials play a critical role in technologies intended to deliver therapeutic agents in clinical settings. Recent explosion of our understanding of how cells utilize nucleic acids has garnered excitement to develop a range of older (e.g., antisense oligonucleotides, plasmid DNA and transposons) and emerging (e.g., short interfering RNA, messenger RNA and non-coding RNAs) nucleic acid agents for therapy of a wide range of diseases. This review will summarize biomaterials-centered advances to undertake effective utilization of nucleic acids for therapeutic purposes. We first review various types of nucleic acids and their unique abilities to deliver a range of clinical outcomes. Using recent advances in T-cell based therapy as a case in point, we summarize various possibilities for utilizing biomaterials to make an impact in this exciting therapeutic intervention technology, with the belief that this modality will serve as a therapeutic paradigm for other types of cellular therapies in the near future. We subsequently focus on contributions of biomaterials in emerging nucleic acid technologies, specifically focusing on the design of intelligent nanoparticles, deployment of mRNA as an alternative to plasmid DNA, long-acting (integrating) expression systems, and in vitro/in vivo expansion of engineered T-cells. We articulate the role of biomaterials in these emerging nucleic acid technologies in order to enhance the clinical impact of nucleic acids in the near future.

Biomaterials for polynucleotide delivery to anchorage-independent cells.

Anchorage-independent cells possess morphological features and cell membrane compositions that are distinct from adherent cells. They display minimal surface area, have a low rate of endocytosis and generally possess few proteoglycans which make it a challenge to deliver nucleic acids into them. Wide ranges of methods and materials have been developed to tackle the delivery obstacles for the polynucleotide-based therapeutics in modifying non-adherent cells. This article summarizes the techniques and biomaterials that have been utilized for transfection of anchorage-independent cells. First, physical techniques are briefly described along with particular applications for which they are well-suited. The structure-activity relationship of various biomaterial carriers of polynucleotides are then discussed with strategies employed to enhance their capability to transfect anchorage-independent cells. In conclusion, the authors' perspectives on different methods for polynucleotide delivery to primary human cells are compared, along with a discussion of their progression towards clinical trials.