Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases.

Hematological disorders result in significant health consequences, and traditional therapies frequently entail adverse reactions without addressing the root cause. A potential solution for hematological disorders characterized by gain-of-function mutations lies in the emergence of small interfering RNA (siRNA) molecules as a therapeutic option. siRNAs are a class of RNA molecules composed of double-stranded RNAs that can degrade specific mRNAs, thereby inhibiting the synthesis of underlying disease proteins. Therapeutic interventions utilizing siRNA can be tailored to selectively target genes implicated in diverse hematological disorders, including sickle cell anemia, ß-thalassemia, and malignancies such as lymphoma, myeloma, and leukemia. The development of efficient siRNA silencers necessitates meticulous contemplation of variables such as the RNA backbone, stability, and specificity. Transportation of siRNA to specific cells poses a significant hurdle, prompting investigations of diverse delivery approaches, including chemically modified forms of siRNA and nanoparticle formulations with various biocompatible carriers. This review delves into the crucial role of siRNA technology in targeting and treating hematological malignancies and disorders. It sheds light on the latest research, development, and clinical trials, detailing how various pharmaceutical approaches leverage siRNA against blood disorders, mainly concentrating on cancers. It outlines the preferred molecular targets and physiological barriers to delivery while emphasizing the growing potential of various therapeutic delivery methods. The need for further research is articulated in the context of overcoming the shortcomings of siRNA in order to enrich discussions around siRNA's role in managing blood disorders and aiding the scientific community in advancing more targeted and effective treatments.

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/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia.

The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro. Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.

Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery.

Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery.

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.

An optimized polymeric delivery system for piggyBac transposition.

The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.

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.

Exploring the Potential of siRNA Delivery in Acute Myeloid Leukemia for Therapeutic Silencing.

We investigated the feasibility of using siRNA therapy for acute myeloid leukemia (AML) by developing macromolecular carriers that facilitated intracellular delivery of siRNA. The carriers were derived from low-molecular-weight (<2 kDa) polyethyleneimine (PEI) and modified with a range of aliphatic lipids. We identified linoleic acid and lauric acid-modified PEI as optimal carriers for siRNA delivery to AML cell lines KG1 and KG1a, as well as AML patient-derived mononuclear cells. As they have been proven to be potent targets in the treatment of AML, we examined the silencing of BCL2L12 and survivin and showed how it leads to the decrease in proliferation of KG1 and stem-cell-like KG1a cells. By optimizing the transfection schedule, we were able to enhance the effect of the siRNAs on proliferation over a period of 10 days. We additionally showed that with proper modifications of PEI, other genes, including MAP2K3, CDC20, and SOD-1, could be targeted to decrease the proliferation of AML cells. Our studies demonstrated the versatility of siRNA delivery with modified PEI to elicit an effect in leukemic cells that are difficult to transfect, offering an alternative to conventional drugs for more precise and targeted treatment options.

Designing Nanomedicines for Breast Cancer Therapy.

In 2020, breast cancer became the most diagnosed cancer worldwide. Conventional chemotherapies have major side effects due to their non-specific activities. Alternatively, short interfering RNA(siRNA)-carrying nanoparticles (NPs) have a high potential to overcome this non-specificity. Lipid-substituted polyethyleneimine (PEI) polymers (lipopolymers) have been reported as efficient non-viral carriers of siRNA. This study aims to engineer novel siRNA/lipopolymer nanocomplexes by incorporating anionic additives to obtain gene silencing through siRNA activity with minimal nonspecific toxicity. We first optimized our polyplexes in GFP+ MDA-MB-231 cells to effectively silence the GFP gene. Inclusion of phosphate buffer with pH 8.0 as complex preparation media and N-Lauroylsarcosine Sodium Salt as additive, achieved ~80% silencing with the least amount of undesired cytotoxicity, which was persistent for at least 6 days. The survivin gene was then selected as a target in MDA-MB-231 cells since there is no strong drug (i.e., small organic molecule) for inhibition of its oncogenic activity. The qRT-PCR, flow cytometry analysis and MTT assay revealed >80% silencing, ~95% cell uptake and >70% cell killing by the same formulation. We conclude that our lipopolymer can be further investigated as a lead non-viral carrier for breast cancer gene therapy.

Nanoparticles and cytokine response.

Synthetic nanoparticles (NPs) are non-viral equivalents of viral gene delivery systems that are actively explored to deliver a spectrum of nucleic acids for diverse range of therapies. The success of the nanoparticulate delivery systems, in the form of efficacy and safety, depends on various factors related to the physicochemical features of the NPs, as well as their ability to remain "stealth" in the host environment. The initial cytokine response upon exposure to nucleic acid bearing NPs is a critical component of the host response and, unless desired, should be minimized to prevent the unintended consequences of NP administration. In this review article, we will summarize the most recent literature on cytokine responses to nanoparticulate delivery systems and identify the main factors affecting this response. The NP features responsible for eliciting the cytokine response are articulated along with other factors related to the mode of therapeutic administration. For diseases arising from altered cytokine pathophysiology, attempts to silence the individual components of cytokine response are summarized in the context of different diseases, and the roles of NP features on this respect are presented. We finish with the authors' perspective on the possibility of engineering NP systems with controlled cytokine responses. This review is intended to sensitize the reader with important issues related to cytokine elicitation of non-viral NPs and the means of controlling them to design improved interventions in the clinical setting.

Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors.

Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (Mw ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.

Potential of siRNA in COVID-19 therapy: Emphasis on in silico design and nanoparticles based delivery.

Small interfering RNA (siRNA)-mediated mRNA degradation approach have imparted its eminence against several difficult-to-treat genetic disorders and other allied diseases. Viral outbreaks and resulting pandemics have repeatedly threatened public health and questioned human preparedness at the forefront of drug design and biomedical readiness. During the recent pandemic caused by the SARS-CoV-2, mRNA-based vaccination strategies have paved the way for a new era of RNA therapeutics. RNA Interference (RNAi) based approach using small interfering RNA may complement clinical management of the COVID-19. RNA Interference approach will primarily work by restricting the synthesis of the proteins required for viral replication, thereby hampering viral cellular entry and trafficking by targeting host as well as protein factors. Despite promising benefits, the stability of small interfering RNA in the physiological environment is of grave concern as well as site-directed targeted delivery and evasion of the immune system require immediate attention. In this regard, nanotechnology offers viable solutions for these challenges. The review highlights the potential of small interfering RNAs targeted toward specific regions of the viral genome and the features of nanoformulations necessary for the entrapment and delivery of small interfering RNAs. In silico design of small interfering RNA for different variants of SARS-CoV-2 has been discussed. Various nanoparticles as promising carriers of small interfering RNAs along with their salient properties, including surface functionalization, are summarized. This review will help tackle the real-world challenges encountered by the in vivo delivery of small interfering RNAs, ensuring a safe, stable, and readily available drug candidate for efficient management of SARS-CoV-2 in the future.

Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers.

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Corrigendum to "The effect of low intensity pulsed ultrasound on mandibular condylar growth in young adult rats" [Bone Rep. 15 (2021) 101122 (December)].

[This corrects the article DOI: 10.1016/j.bonr.2021.101122.].

siRNA Functionalized Lipid Nanoparticles (LNPs) in Management of Diseases.

RNAi (RNA interference)-based technology is emerging as a versatile tool which has been widely utilized in the treatment of various diseases. siRNA can alter gene expression by binding to the target mRNA and thereby inhibiting its translation. This remarkable potential of siRNA makes it a useful candidate, and it has been successively used in the treatment of diseases, including cancer. However, certain properties of siRNA such as its large size and susceptibility to degradation by RNases are major drawbacks of using this technology at the broader scale. To overcome these challenges, there is a requirement for versatile tools for safe and efficient delivery of siRNA to its target site. Lipid nanoparticles (LNPs) have been extensively explored to this end, and this paper reviews different types of LNPs, namely liposomes, solid lipid NPs, nanostructured lipid carriers, and nanoemulsions, to highlight this delivery mode. The materials and methods of preparation of the LNPs have been described here, and pertinent physicochemical properties such as particle size, surface charge, surface modifications, and PEGylation in enhancing the delivery performance (stability and specificity) have been summarized. We have discussed in detail various challenges facing LNPs and various strategies to overcome biological barriers to undertake the safe delivery of siRNA to a target site. We additionally highlighted representative therapeutic applications of LNP formulations with siRNA that may offer unique therapeutic benefits in such wide areas as acute myeloid leukaemia, breast cancer, liver disease, hepatitis B and COVID-19 as recent examples.

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.

In Vitro Cytotoxicity and Cytokine Production by Lipid-Substituted Low Molecular Weight Branched PEIs Used for Gene Delivery.

Lipid-modified low molecular weight branched polyethyleneimines (PEIs) are promising non-viral gene delivery systems that have been successfully explored for treatment of various diseases. The present study aims to determine in vitro safety of these delivery systems based on assessment of cytotoxicity with peripheral blood mononuclear cells (PBMCs), hemolysis with human red blood cells (RBC) and cytokine secretion from several sources of PBMCs. The viability of cells treated with lipopolymer/pDNA complexes was dependent on the polymer:pDNA ratio used but remained low at therapeutically relevant concentrations for most lipopolymers, except for the propionic acid substituted PEIs. The extent of hemolysis was minimal and below the accepted safety levels with most of the lipopolymers; however, some linoleic acid substituted PEIs yielded significant hemolysis activity. Unlike strong cytokine secretion from PMA/IO stimulated cells, most lipopolymer/pDNA complexes remained non-responsive, showing minimal changes in cytokine secretion (TNF-α, IL-6 and IFN-γ) irrespective of the lipopolymer/pDNA formulations. The 0.6 kDa PEI with lauric acid substituent displayed slight cytokine upregulation, however it remained low relative to the positive controls. This study demonstrated that the lipid modified LMW PEIs are expected to be safe in contact with blood components. However, close attention to lipopolymer concentration and ratio of polymer to pDNA in formulations might be required for individual lipopolymers for optimal safety response in nucleic acid therapies. STATEMENT OF SIGNIFICANCE: This manuscript investigated the safety aspects of various lipid modified low molecular weight polyethylenimine (LMW-PEI) polymers employed for pDNA delivery through in vitro studies. Using peripheral blood mononuclear cells (PBMCs) from multiple sources, we show that the hemolysis ability was minimal for most polymers, although a particular lipid substituent (linoleic acid) at specific ratios exhibited hemolysis. The levels of pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ) were slightly upregulated only with a lauric acid substituted 0.6PEI, but remained low relative to positive control treatments. We further report the beneficial effect of polyacrylic acid additives on hemolysis and cytokine secretion to a reasonable extent. This study confirms the feasibility of using LMW-PEI as safe delivery agents for various therapeutic purposes.