Cytotoxicity Of Polyethylenimine Research Articles

Self-Adjuvanting Polyguanidine Nanovaccines for Cancer Immunotherapy.

Tapping into the innate immune system's power, nanovaccines can induce tumor-specific immune responses, which is a promising strategy in cancer immunotherapy. However, traditional vaccine design, requiring simultaneous loading of antigens and adjuvants, is complex and poses challenges for mass production. Here, we developed a tumor nanovaccine platform that integrates adjuvant functions into the delivery vehicle, using branched polyguanidine (PolyGu) nanovaccines. These nanovaccines were produced by modifying polyethylenimine (PEI) with various guanidine groups, transforming PEI's cytotoxicity into innate immune activation. The PolyGu nanovaccines based on poly(phenyl biguanidine ) (Poly-PBG) effectively stimulated dendritic cells, promoted their maturation via the TLR4 and NLRP3 pathways, and displayed robust in vivo immune activity. They significantly inhibited tumor growth and extended mouse survival. The PolyGu also showed promise for constructing more potent mRNA-based nanovaccines, offering a platform for personalized cancer vaccine. This work advances cancer immunotherapy toward potential clinical application by introducing a paradigm for developing self-adjuvanting nanovaccines.

Lipid-Polymer Hybrid Nanoparticles with Both PD-L1 Knockdown and Mild Photothermal Effect for Tumor Photothermal Immunotherapy.

In developing countries, the incidence of colorectal cancer (CRC) is on the rise. The combination of programmed cell death ligand-1 (PD-L1) siRNA (siPD-L1) and mild photothermal therapy (PTT) is a promising strategy for CRC treatment. In this study, dopamine-modified polyethylenimine (PEI) was prepared to fabricate an IR780 and siPD-L1 codelivery lipid-polymer hybrid nanoparticle (lip@PSD-siP) for the photothermal immunotherapy of CRC. The modification of dopamine can significantly reduce the cytotoxicity of PEI. lip@PSD-siP can be effectively taken up by CT26 cells and successfully escaped from lysosomes after entering the cells for 4 h. After CT26 cells were transfected with lip@PSD-siP, the PD-L1 positive cell rate decreased by 82.4%, and its PD-L1 knockdown effect was significantly stronger than the positive control Lipo3000-siP. In vivo studies showed that lip@PSD-siP-mediated mild PTT and efficient PD-L1 knockdown exhibited primary and distal tumor inhibition, metastasis delay, and rechallenged tumor inhibition. The treatment with lip@PSD-siP significantly promoted the maturation of dendritic cells in lymph nodes. The amount of T cell infiltration in the tumor tissues increased significantly, and the frequency of CD8+ T cells and CD4+ T cells was significantly higher than that of other groups. The percentage of immunosuppressive regulatory cells (Tregs) in the tumor tissue on the treatment side decreased by 88% compared to the PBS group, and the proportion of CD8+CD69+ T cells in the distal tumor tissue was 2.8 times that of the PBS group. The memory T cells of mice in the long-term antitumor model were analyzed. The results showed that after treatment with lip@PSD-siP, the frequency of effector memory T cells (Tem cells) significantly increased, suggesting the formation of immune memory.

Reduced cytotoxicity of polyethyleneimine by covalent modification of antioxidant and its application to microalgal transformation

ABSTRACT The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation.

An Efficient and Inexpensive PEI-mediated Transfection for Transient Overexpression of CYP2C9 in WRL 68 Normal Liver Cells

Aims: This study aims to develop an efficient, and inexpensive transfection procedure using low cost polyethylenimine (PEI) as transfection reagent for overexpression of CYP2C9 in WRL 68 host cells.
 Introduction: Overexpression of CYP450 isozymes such as CYP2C9 has been a reliable approach in developing a high specificity in vitro tool for inhibition study of this liver enzyme. Transient or stable transfection are used to insert plasmid DNA into a mammalian host cell in order to produce proteins. There are many efficient transfection reagents available in the market for this purpose, however, it can be expensive, and a low-cost alternative is favorable.
 Methodology: Cytotoxicity of PEI was screened on four cell lines namely WRL 68, HepG2, MCF-7 and A549 using MTS assay. WRL 68 was transiently transfected with CYP2C9 plasmid using PEI and DNA concentration as well as transfection time was optimized for the best efficiency and expression. Expression of CYP2C9 protein was measured using qPCR and further evaluated with western blot analysis.
 Results: IC50 values of PEI are 0.327 ± 0.013 mg/mL (WRL-68), 0.395 ± 0.037 mg/mL (HepG2), 1.159 ± 0.032 mg/mL (A549) and 1.281 ± 0.000 mg/mL (MCF-7). Combination of 3ug plasmid DNA with 2.8 μM of PEI and 10.5 mM NaCl resulted in the highest transfection efficiency and expression after 48 hours.
 Conclusion: A low-cost and efficient PEI transient transfection procedure was optimized for CYP2C9 overexpression and useful for the purpose of further development of cell-based enzyme inhibition model.

Low molecular weight polyethyleneimine modified by 2-aminoimidazole achieving excellent gene transfection efficiency

The transfection efficiency and cytotoxicity of polyethyleneimine (PEI), as a non-viral vector, increase with molecular weight. Low molecular weight PEI, such as branched PEI1800 (Mw ~ 1800 kDa), has negligible transfection efficiency but low cytotoxicity. Given the low efficiency of PEI1800, a variety of modifications were explored. A structure-integrated group of guanidyl and imidazolyl, 2-aminoimidazole was designed (Scheme 1). To verify the molecular interaction between 2-aminoimidazole and gene, molecular docking experiment, 1H NMR, FTIR and isothermal titration calorimetry (ITC) measurement were carried out. Studies confirmed that 2-aminoimidazole form double hydrogen bonds with phosphate group, which enhanced the thermodynamic interaction between 2-aminoimidazole and gene. Hence, we synthesized 2-aminoimidazole modified PEI1800, and as controls, guanidyl and imidazolyl modified PEI1800 were synthesized respectively. As expected, the results showed that 2-aminoimidazole modified PEI1800 could condense DNA into a higher positively charged complex with 116 ~ 120 nm in diameter and improve the cellular uptake. EGFP assay and luciferase assay showed that 2-aminoimidazole modified PEI1800 exhibited much better transfection than PEI1800, guanidyl modified PEI1800, imidazolyl modified PEI1800 both in HEK 293 T cells and SGC-7901 cells. Besides, MTT assay showed 2-aminoimidazole modified PEI1800 exhibited better biocompatibility than PEI25 K. These results highlight the critical role that 2-aminoimidazole played in non-viral gene delivery.

Impact of oppositely charged shell and cores on interaction of core-shell colloids with differently charged proteins as a route for tuning of the colloids cytotoxicity

The present work represents interactions between the core-shell nanoparticles and different proteins, exemplified by lysozyme (LSZ), pepsin, bovine serum albumin (BSA), thioredoxin (TRX) and yellow fluorescent protein (YFP). The core-shell morphology derives from the non-covalent deposition of polyethyleneimine (PEI) onto nanoprecipitated luminescent complex (AuCl)2L (L is cyclic PNNP ligand). Analysis of the data obtained by DLS, CD spectroscopy, luminescence derived from both (AuCl)2L and YFP reveal the electrostatically driven interaction of negatively charged proteins with the shell of PEI-(AuCl)2L. The fluorescence of YFP enables to reveal the inclusion of the protein molecules into the shell. The lack of any luminescent response of PEI-(AuCl)2L on TRX conforms its electrostatically driven interactions with the shell which, in turn, excludes a binding of the exposed thiol moieties with (AuCl)2L. The negatively charged surface of pepsin provides the greatest recharging of the PEI-based shell versus the other proteins, which is followed by the enhanced luminescence of (AuCl)2L. The significant effect of PEI-(AuCl)2L on the CD spectra of LSZ followed by the decreased intensity of (AuCl)2L-based luminescence points to specific interaction mode of PEI-(AuCl)2L with LSZ. The flow cytometry and fluorescent microscopy measurements revealed efficient internalization of PEI-(AuCl)2L into the Wi-38 cell samples resulting in the efficient staining of all cell organelles. The concentration dependent cytotoxicity of PEI-(AuCl)2L is detectably enhanced by LSZ, which is correlated with its interaction mode with the nanoparticles.

PEI fluorination reduces toxicity and promotes liver-targeted siRNA delivery.

Polyethyleneimine (PEI) has been extensively investigated as an efficient carrier for nucleic acid delivery. Yet, it suffers from a high toxicity profile that hinders clinical translation. Fluorination has proven to be a valid approach to reduce the cytotoxicity of PEI and improve the in vitro siRNA delivery potency. Hydrophobicity and lipophobicity can be controllably introduced into the side chains of PEI. However, the effect of fluorination on siRNA delivery in vivo, particularly the biodistribution of siRNA polyplex nanoparticles with fluorinated PEIs, has not been extensively explored. Here, we introduce two series of fluorinated PEIs via amidation with ethyl trifluoroacetate and perfluorobutyryl chloride. Fluorination substantially improved the performance of PEI for siRNA delivery by reducing the cytotoxicity to MDA-MB-231 cells. Importantly, fluorinated PEI enabled the major accumulation of siRNA polyplex nanoparticles in the liver while non-fluorinated PEI delivered siRNA nanoparticles mainly to the lungs after intravenous administration to mice. It is envisioned that fluorination may be an important general strategy for lowering toxicity of cationic polymers, and that the fluorination-induced alteration of biodistribution may be applicable for improved delivery to different organs. Graphical abstract.

Transfection efficiency and cytotoxicity of polyethyleneimine-coated magnetic iron oxide nanoparticles in rooster sperm cells.

Since the morphology of the rooster spermatozoa is different to other animal spermatozoa, the aim of the current study was to investigate the transfection efficiency and cytotoxicity of polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (MION) on these cells. Liposome/nucleic acid (NA) complexes and PEI-coated MION linked to the labeled oligonucleotides were used. Viability and percentage of exogenous nucleic acid uptake of spermatozoa were measured by flow cytometry analyses. The results showed a significant increase in exogenous nucleic acid uptake by rooster spermatozoa (P < 0.001) when treated with MION-NA complexes in comparison to liposome/NA. There were no significant differences between efficiency of lipid-based transfection agent and MION (P > 0.05) during short incubation period. MION with or without magnetic field, did not show significant cytotoxicity while the lipid-based transfection agent significantly decreased (P < 0.05) the viability of rooster spermatozoa. Results of this study showed that magnetofection and lipofection were both effective methods which increased exogenous nucleic acid uptake by rooster spermatozoa. However, the magnetofection method was more successful in maintaining the cell's survival than lipofection method.

Surface Charge Modification of Polyethyleneimine for Enhanced Renal Clearance and Bioimaging

Polyethyleneimine (PEI) has emerged as a potentially effective carrier for gene delivery. To overcome the cytotoxicity of PEI in vivo, the biodistribution and clearance of PEI conjugated with near-infrared fluorophores (NIRF) were imaged in mice. To confirm the effect of surface charge on enhanced renal clearance, PEI was conjugated with three different types of NIRF: positively charged PEI-NIRF+, negatively charged PEI-NIRF-, and zwitterionic PEI-NIRF± conjugates, which were intravenously injected into mice. The biodistribution and body clearance were monitored under an NIR fluorescence imaging system at 4 h and 24 h postinjection. All organs and tissues were resected to further compare the distribution of each PEI-NIRF conjugate in two mice at 4 h and 24 h. The results showed that the zwitterionic PEI-NIRF± conjugate was most rapidly cleared, primarily through the kidneys of the mice within 24 h postinjection. In conclusion, the biodistribution of zwitterionic PEI-NIRF± was found to be significantly different from that of positively and negatively charged PEI-NIRF conjugates with increased renal clearance. Thus, zwitterionic PEI-NIRF± could be a promising gene delivery carrier that can potentially reduce the side effects of conventional cationic polymers such as cytotoxicity and non-specific binding in vivo.

LB1551 How to balance the adjuvanticity and cytotoxicity of polyethylenimine as melanoma vaccines

LB1551 How to balance the adjuvanticity and cytotoxicity of polyethylenimine as melanoma vaccines

Rational Surface Design of Upconversion Nanoparticles with Polyethylenimine Coating for Biomedical Applications: Better Safe than Brighter?

Upconversion nanoparticles (UCNPs) coated with polyethylenimine (PEI) are popular background-free optical contrast probes and efficient drug and gene delivery agents attracting attention in science, industry, and medicine. Their unique optical properties are especially useful for subsurface nanotheranostics applications, in particular, in skin. However, high cytotoxicity of PEI limits safe use of UCNP@PEI, and this represents a major barrier for clinical translation of UCNP@PEI-based technologies. Our study aims to address this problem by exploring additional surface modifications to UCNP@PEI to create less toxic and functional nanotheranostic materials. We designed and synthesized six types of layered polymer coatings that envelop the original UCNP@PEI surface, five of which reduced the cytotoxicity to human skin keratinocytes under acute (24 h) and subacute (120 h) exposure. In parallel, we examined the photoluminescence spectra and lifetime of the surface-modified UCNP@PEI. To quantify their brightness, we developed original methodology to precisely measure the colloidal concentration to normalize the photoluminescence signal using a nondigesting mass spectrometry protocol. Our results, specified for the individual coatings, show that, despite decreasing the cytotoxicity, the external polymer coatings of UCNP@PEI quench the upconversion photoluminescence in biologically relevant aqueous environments. This trade-off between cytotoxicity and brightness for surface-coated UCNPs emphasizes the need for the combined assessment of the viability of normal cells exposed to the nanoparticles and the photophysical properties of postmodification UCNPs. We present an optimized methodology for rational surface design of UCNP@PEI in biologically relevant conditions, which is essential to facilitate the translation of such nanoparticles to the clinical applications.

Enhancement of the transfection efficiency of DNA into Crocus sativus L. cells via PEI nanoparticles

Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine (PEI) was used for gene therapy successfully. The present study investigated the effect of PEI and ultrasound waves on ssDNA delivery into saffron cells. Gel retardation, dynamic light scattering (DLS) and scanning electron microscopy (SEM) assays were employed to determine the physicochemical properties of PEI/f-DNA polyplex (complex of PEI and fluorescently labeled DNA). Moreover, the cytotoxicity of PEI, PEI/f-DNA polyplex and ultrasound were investigated on saffron cells at different concentrations. The gel retardation results indicated that the formation and neutralization of the PEI/f-DNA polyplex were completed at N/P=5. The particle size distribution of the polyplexes was from 50 to 122 nm. The experimental results revealed that the cytotoxicity of the PEI/f-DNA polyplex was lower than that of PEI alone, hence the cells showed both dose- and exposure duration-dependent responses. Furthermore, the viability of saffron cells declined extremely after 5 and 10 min sonication but this reduction was not significant at 2 min exposure duration. The results also indicated that the combined utilization of ultrasound and PEI nanoparticles increased the transfection efficiency of saffron cells up to two times higher than those obtained by PEI or ultrasound separately.

Macrocycle-wrapped polyethylenimine for gene delivery with reduced cytotoxicity.

Due to its outstanding capability to facilitate DNA condensation, transportation and endosomal escape, polyethylenimine (PEI) has been frequently studied for gene delivery. However, its molecular weight (M.W.) dependent transfection efficiency and cytotoxicity has severely limited its clinical application. To resolve this dilemma, a supramolecular strategy was developed for the first time, in which PEI with large M.W. (branched, 25 kDa) that has a satisfactory transfection efficiency, yet high non-specific cytotoxicity for gene delivery was wrapped with macrocyclic cucurbit[7]uril (CB[7]). The successful wrapping of the PEI by the macrocyclic CB[7] was proved by 1H NMR spectroscopy and supported by isothermal titration calorimetry (ITC). The plasmid DNA (pDNA) condensability of PEI was not affected by the supramolecular coating as evidenced from the agarose gel electrophoresis assay. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results demonstrated that the particle size, zeta potential, and morphology of the self-assemblies of PEI/pDNA and PEI/CB[7]/pDNA were comparable. As a consequence of the supramolecular wrapping, the cytotoxicity of PEI was significantly constrained as demonstrated by MTT assay, apoptosis assay, and a hemolysis study. In particular, both the cellular uptake and the gene transfection efficiency results suggest that the supramolecular wrapping of PEI by CB[7] exhibits negligible effects on PEI, thus functioning as an effective non-viral gene delivery vector. This novel supramolecular-wrapping strategy provides new insights for facile alleviation of the non-specific toxicity of PEI and potentially other polycationic gene vectors without compromising their transfection efficiency.

Conformation and activity alteration of horseradish peroxidase induced by the interaction with gene carrier polyethyleneimines

Polyethyleneimine (PEI) has long been considered as “golden standard” for polymeric gene delivery carriers. However the molecular basis of the cytotoxicity of PEI is poorly understood. Little is known about the effects of PEI on the structure and functions of biomacromolecules. In this work, fluorescence, UV–vis absorption, circular dichroism spectroscopy were conducted to investigate the influence of PEI of average molecular weight 25, 10 and 1.8kDa (denoted as PEI25k, PEI10k and PEI1.8k) on the conformation of horseradish peroxidase (HRP) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the mechanism of the interaction between PEIs and HRP. PEIs were found to bind onto the surface of HRP predominantly via hydrophobic interaction and hydrogen bond or van der Waals interaction. The complex formation between HRP and PEI induced a more compact conformation of the enzyme and an increased hydrophobicity of the microenvironment surrounding heme pocket. The conformational change of HRP had little impact on the affinity towards H2O2 and phenol. However, the increase in the non-planarity of porphyrin ring in the heme group led to an increase in the exposure degree of the active center and thus an enhancement of catalytic efficiency of HRP in the presence of high molecular weight PEIs (PEI25k and PEI10k). The polymer size played an important role in PEI-HRP interaction. PEI of low molecular weight (PEI1.8k) was less efficient to alter the conformation and catalytic activity of HRP in aqueous solutions.

Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in CHO cells

Polyethyleneimine (PEI) is a polycationic compound frequently used as a transfection agent. However, cytotoxicity of PEI and PEI-coated nanoparticles (PEI NPs) is still a major obstacle in its use. In this study we report a method for reducing cytotoxicity of PEI NPs by addition of glutathione in NPs synthesis. Glutathione reduced cytotoxic effects for at least 30% and decreased observed oxidative stress response compared to standard formulation. Results showed that the effect was partially due to reduced zeta potential and partially due to protective antioxidant properties of glutathione. Addition of glutathione to cell culture media with concurrent exposure to PEI NPs proved to be insufficient for cytotoxicity reduction. Additionally, we compared internalization pathways of both PEI NPs and GSH NPs. NPs were only found in endosomes and no NPs were found free in the cytosol, as would be expected according to so called proton sponge hypothesis.

The effects of polyethylenimine/DNA nanoparticle on transcript levels of apoptosis-related genes

Context: Polyethylenimine (PEI) is a cationic polymer commonly used in gene transfer. Although numerous investigations have indicated that PEI can induce apoptosis/necrosis but the mechanism of its cytotoxicity is still poorly understood. Objective: The purpose of this study was to investigate the effects of PEI/DNA complexes on the expression of apoptotic genes in human colon adenocarcinoma cells (HT29). Methods: HT29 cells were exposed to PEI/DNA complex (C/P = 0.8) for 24 h. Then, qRT PCR was used to assess the expression of 26 apoptotic-related genes. Result: Analysis of the transcript level of genes revealed that while the expression of anti-apoptotic genes such as Bclx, Bcl2, NFkB, and AIF was not significantly reduced but the expression of pro-apoptotic genes such as Fasl, Bax, TNFR1, DR4, Casp8, and cytochrome C was considerably increased in transfected HT29 cell lines. Conclusions: Our results showed that PEI could increase the level of pro-apoptotic genes and decrease antiapoptotic genes as a possible mechanism involved in PEI cytotoxicity.

Grafting polyethylenimine with quinoline derivatives for targeted imaging of intracellular Zn2 + and logic gate operations

In this study, a highly sensitive and selective fluorescent Zn2+ probe which exhibited excellent biocompatibility, water solubility, and cell-membrane permeability, was facilely synthesized in a single step by grafting polyethyleneimine (PEI) with quinoline derivatives. The primary amino groups in the branched PEI can increase water solubility and cell permeability of the probe PEIQ, while quinoline derivatives can specifically recognize Zn2+ and reduce the potential cytotoxicity of PEI. Basing on fluorescence off-on mechanism, PEIQ demonstrated excellent sensing capability towards Zn2+ in absolute aqueous solution, where a high sensitivity with a detection limit as low as 38.1nM, and a high selectivity over competing metal ions and potential interfering amino acids, were achieved. Inspired by these results, elementary logic operations (YES, NOT and INHIBIT) have been constructed by employing PEIQ as the gate while Zn2+ and EDTA as chemical inputs. Together with the low cytotoxicity and good cell-permeability, the practical application of PEIQ in living cell imaging was satisfactorily demonstrated, emphasizing its wide application in fundamental biology research.

Gene transfection of human mesenchymal stem cells with anano-hydroxyapatite-collagen scaffold containing DNA-functionalized calcium phosphate nanoparticles.

This study aimed to fabricate a growth factor-releasing biodegradable scaffold for tissue regeneration. We prepared multishell calcium phosphate (CaP) nanoparticles functionalized with DNA, polyethyleneimine (PEI), protamine and octa-arginine (R8) and compared their respective transfection activity and cell viability measures using human mesenchymal stem cells. DNA-protamine complexes improved the transfection efficiency of CaP nanoparticles with the exception of those functionalized with R8. These complexes also greatly reduced the cytotoxicity of PEI. In addition, we also fabricated DNA-protamine-functionalized CaP nanoparticle-loaded nano-hydroxyapatite-collagen scaffolds and investigated their gene transfection efficiencies. These experiments showed that the scaffolds were associated with moderate hMSC cell viability and were capable of releasing the BMP-2 protein into hMSCs following gene transfection. In particular, the scaffold loaded with protamine-containing CaP nanoparticles showed the highest cell viability and transfection efficiency in hMSCs; thus, it might be suitable to serve as an efficient growth factor-releasing scaffold.

Attaching Biosynthesized Bacterial Magnetic Particles to Polyethylenimine Enhances Gene Delivery Into Mammalian Cells.

Gene transfection using bacterial magnetic particles (BMPs)-polyethylenimine (PEI) has become increasingly prevalent; however, relatively little effort has been made to optimize the protocol for preparing these complexes with the aim of improving their transfection efficiency. Here, we report a procedure for constructing BMPs-PEI/DNA complexes that results in improved transfection efficiency, reduced cytotoxicity and shorter procedure times for both complex formation and transfection over current methods. BMPs-PEI/DNA complexes mixed using ultrasonication yielded beads that were 10.2% more efficient at transfecting HeLa cells than complexes made by mechanical vortexing. Phosphate-buffered saline (PBS) proved to be a superior solvent for BMPs-PEI/DNA and PEI/DNA complexes, and the transfection efficiencies in HeLa cells were 54.75% and 46.01%, respectively. Comparable levels of transfection were achieved after 10 min of incubation with low-dose BMPs-PEI/DNA complexes versus 4 h with standard PEI/DNA complexes. BMPs stored in PBS have an average transfection efficiency that is 5% greater than those stored in physiological salt solutions. Cell morphology and cytotoxicity analyses demonstrated that the biosynthesized BMPs lessened the cytotoxicity of PEI to cells. Our results provide an optimized protocol for BMPs-PEI/DNA complex construction and gene transfer in vitro.

Silica-collagen nanocomposites for local and sustained release of Interleukin 10 (IL-10) to treat cutaneous chronic wounds

Event Abstract Back to Event Silica-collagen nanocomposites for local and sustained release of Interleukin 10 (IL-10) to treat cutaneous chronic wounds Xiaolin Wang1, Thibaud Coradin1 and Christophe Helary1 1 Sorbonne Universités, UPMC Univ Paris 06, CNRS, Collège de France, UMR 7574, Chimie de la Matière Condensée, France Cutaneous chronic wounds are characterized by the absence of healing six weeks after the injury. The classic treatment is the debridement of the wound bed followed by a compression method. When this technique is not effective enough, the application of wound dressings is required. Several hulman trials with protein therapy have failed because of the rapid diffusion and short half-life of the cytokines in the wound[1]. Therefore gene therapy represents an interesting alternative. Local delivery of nucleic acids through a hydrogel extends the application of gene therapy for the treatment of chronic wounds. However, the inactivation and fast release of encapsulated DNA strongly compromise their efficacy. Nanoscale delivery systems such as silica nanoparticles (SiNPs) hold great promise for gene therapy owing to their high cellular uptake, DNA protection and possible cell targeting[2]. In this study, nanocomposites consisting of plasmid DNA-PEI-SiNP complexes, collagen hydrogel and 3T3 mouse fibroblasts have been evaluated as gene delivery systems to obtain a local and sustained release of biomolecules. Plasmid DNA encoding for Interleukin 10 (IL-10) was chosen as this cytokine is able to modulate inflammation of chronic wounds. SiNPs were modified with PEI (polyethyleneimine) of different molecular weights (1.8 kDa, 10 kDa and 25 kDa) by electrostatic absorption. SiNP-PEI complexes were associated with plasmids encoding for Luciferase or human IL-10 at a weight ratio of 30:1. Then, complexes were encapsulated within collagen hydrogel with 3T3 mouse fibroblasts. Cell transfection was assessed by measuring the luciferase activity or the IL-10 production. Cell viability was evaluated by Alamar Blue Assay. Subsequently, the effect of nanocomposites to modulate inflammation was evaluated on activated mouse RAW 264.7 macrophages. The ability of IL-10 doses released from nanocomposites to downregulate the TNF-α expression in macrophages was analyzed over one week. Successful cell transfection within the collagen/silica nanocomposites was detected by sustained release of luciferase over one week. The best transfection efficiency was obtained with PEI 10KDa (Figure 1). When compared with the soluble form of PEI 25 KDa, the transfection was lower. However, the incubation with increasing quantities of soluble PEI or complexed with SINP revealed a cytotoxicity of PEI 25 KDa and 10 KDa from 10 µg/mL (Figure 1). In contrast, PEI was not toxic until the 100 µg/mL when associated with silica nanoparticles. Interestingly, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Finally, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Tested on activated macrophages, nanocomposites permitted the sustained release of IL-10 (500 pg/mL) which could inhibit the expression of TNF-α by 50% in macrophages (Figure 2). These results show that nanocomposites obtained by entrapping SiNP-PEI-DNA together with cells inside a collagen hydrogel can work as a reservoir for local and controlled gene delivery. In addition, it is possible to create cell factories producing high doses of IL-10 which modulate inflammation. These biomaterials are promising for the local treatment of cutaneous chronic wounds. Bernard Haye; Corinne Illoul