The effect of novel tyrosine-modified polyethyleneimines on human albumin structure - Thermodynamic and spectroscopic study.

The interaction of proteins with nanoparticle components are crucial for the evaluation of nanoparticle function, toxicity and biodistribution. Polyethyleneimines (PEIs) with defined tyrosine modifications are a class of novel polymers designed for improved siRNA delivery. Their interactions with biomacromolecules are still poorly described. This paper analyzes the interaction of different tyrosine-modified PEIs with human serum albumin as the most abundant serum protein. The ability of tyrosine modified, linear or branched PEIs to bind human serum albumin (HSA) was analyzed and further characterized. The interaction with hydrophobic parts of protein were studied using 1- nilinonaphthalene-8-sulfonic acid (ANS) and changes in the HSA secondary structure were evaluated using circular dichroism (CD). Complex formation and sizes were studied by transmission electron microscopy (TEM) and dynamic light scattering methods (DLS). We demonstrate that tyrosine modified PEIs are able to bind human serum albumin. Based on thermodynamic studies, van der Waals interaction, H-bonding and hydrophobic interactions are determined as main molecular forces involved in complex formation. Analysis of secondary structures revealed that the polymers decreased α-helix content, while increasing levels of randomly folded structures. Complex formation was confirmed by TEM and DLS. These findings are crucial for understanding polymer-protein interactions and the properties of nanoparticles.

Polyethylenimine linked with chitosan improves astaxanthin production in Haematococcus pluvialis.

Astaxanthin is receiving increasing interest as an antioxidant and high value-added secondary metabolite. Haematococcus pluvialis is the main source for astaxanthin production, and many studies are being conducted to increase the production of astaxanthin. In this study, we linked polyethylenimine (PEI) with chitosan to maintain astaxanthin-inducing ability while securing the recyclability of the inducer. Astaxanthin accumulation in H. pluvialis was induced to 86.4 pg cell-1 with the PEI-chitosan fiber (PCF) treatment prepared by cross-linking of 10 µM PEI and low molecular weight (MW) chitosan via epichlorohydrin. PEI concentration affected the astaxanthin accumulation, whereas the MW of chitosan did not. In addition, the PCF treatment in H. pluvialis increased the reactive oxygen species (ROS) content in cells, thereby upregulating the transcription of enzymes involved in astaxanthin biosynthesis. PCF can be reused multiple times with the maintenance of over 90% of the astaxanthin production efficiency. This study offers a reusable PCF stimulation strategy for enhancing natural astaxanthin content, and PCF treatment will easily increase the production scale or reduce production costs by using recyclability that is not available in current methods. KEY POINTS: • Polyethylenimine-chitosan fiber (PCF) was applied to Haematococcus pluvialis • PCF promotes astaxanthin accumulation by enhancing oxidative stress in H. pluvialis • PCF can be reused multiple times with maintaining over 90% production efficiency.

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.

Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.

Exosome-Functionalized Ti6Al4V Scaffolds Promoting Osseointegration by Modulating Endogenous Osteogenesis and Osteoimmunity.

Periprosthetic bone defects are the most serious problem of revision total hip arthroplasty, which can easily lead to insufficient osteointegration between the prosthesis and host bone. Bone marrow mesenchymal stem cells (BMSCs) and a moderate inflammatory response at the prosthesis-bone interface play an important role in osteointegration. Here, we developed microarc oxide titanium implant loaded engineered exosomes (S-Exos) to promote osseointegration at the prosthesis-bone interface. First, Smurf1-shRNA was transferred into the BMSCs using a viral vector to prepare S-Exos, which were subsequently immobilized to the microarc oxide titanium implant surface with positively charged polyethyleneimine. The immobilized S-Exos could be slowly and uniformly released and subsequently phagocytosed by BMSCs and macrophages. Once the S-Exos were phagocytosed, they could simultaneously activate the BMP/Smad signaling pathway in the BMSCs and promote macrophage M2 polarization, both of which enhance osseointegration. Specifically, this S-Exos coating exhibits a dual effect of promoting osseointegration, including the osseointegration of BMSCs by activating the BMP/Smad signaling pathway and the macrophage M2 polarization promoting osseointegration. In summary, the construction of S-Exos modified microarc oxide titanium implants could provide a new method for promoting osteointegration between the prosthesis and host bone in revision total hip arthroplasty.

Anhydride chemistry-based hexanoylation of polyethylenimine increases transfection efficiency and expression of tagged DNA for therapeutic proteins in cultured cells.

Transfection of nucleic acid molecules into mammalian cells can be facilitated using viral vectors, electroporation, or biocompatible cationic materials. However, safety issues and the requirement of specialized equipment limits the use of viral vectors and physical methods of transfection like electroporation and microinjection, respectively. Biocompatible cationic lipids and polymers like branched-polyethyleneimine (bPEI) have a wide transfection range and are user-friendly in most applications. However, bPEI exhibits low transfection efficiency in most cell types. In the present work, we have crosslinked the hexanoyl group to bPEI using anhydride chemistry to enhance its efficiency as a transfection reagent. The efficient association of hexanoyl group to bPEI was assessed using Fourier-transform infrared spectroscopy and other physicochemical methods. Hexanoyl-modified bPEI (FA6-bPEI) was found to exhibit significantly enhanced transfection efficiency in both cell lines and cultured primary cells, as compared to native bPEI and the commercially available transfection reagent, Lipofectamine 3000. Furthermore, our in vitro studies indicated that FA6-bPEI can be used for robust transfection for increased production of therapeutic proteins in a cell culture-based system. These results suggested that hexanoyl-modified bPEI can serve as an efficient transfection reagent for studies on hard-to-transfect cells and for enhanced production of therapeutic proteins in vitro.

ROS responsive polyethylenimine-based fluorinated polymers for enhanced transfection efficiency and lower cytotoxicity.

Cationic polymer polyethylenimine (PEI) plays a crucial role in gene delivery. However, high molecular weight PEI leads to higher efficient transfection efficacy and higher cytotoxicity while low molecular weight PEI exhibits lower transfection performance with lower toxicity. Therefore, effective chemical modification of PEI is required to enhance transfection activity and improve biocompatibility. Here, reactive oxygen species (ROS) responsive PEI-based fluorinated polymers (TKPF) with three degrees of fluorination (TKPF12.5%, TKPF25% and TKPF50%) were designed and synthesized by crosslinking low molecular weight PEI (PEI 1.8K) with a thioketal (TK) linker and then modifying heptafluorobutyric anhydride onto their surface. Such gene vectors exhibited the following features: (1) fluorination reduced the positive charge density and endowed hydrophobic and lipophobic characteristics to resist serum interactions; (2) The fluorophilic effect mediated efficient cellular uptake and endosomal escape; (3) ROS-responsive TK linker allowed the polyplex disassembly to decrease the cytotoxicity of the polycations and improve the release of payloads at specific sites. TKPFs attained superior transfection efficiency in multiple cell lines (293TN cells and B16F10 cells) in vitro and showed excellent biocompatibility. Notably, TKPFs also exhibited great serum resistance in gene delivery and TKPF50% transfected nearly 80% cells in the presence of 70% FBS. These results demonstrates that the fluorination and ROS responsiveness combined polycations are excellent gene-delivery vectors with serum-resistant capacity for further application.

Gene delivery of chitosan-graft-polyethyleneimine vectors loaded on scaffolds for nerve regeneration.

As an important transcription factor, c-Jun could upregulate growth factors expression in Schwann cells (SCs). Arginine-Glycine-Aspartate (RGD)-functionalized chitosan-graft-polyethyleneimine (RCP) gene vectors were prepared through the maleic anhydride & the carbodiimide methods, and electrostatically bound with c-Jun plasmids (pJUN), finally loaded on poly-L-lactic acid/silk fibroin parallel fiber films to fabricate nerve scaffold (RCP/pJUN-PSPF@PGA), which could locally deliver c-Jun plasmids into SCs via the mediation of RGD peptides, and upregulate the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in SCs. After the scaffold was bridged in sciatic nerve defect, the delivery of c-Jun plasmids from RCP/pJUN-PSPF@PGA facilitated SCs to sustain the expressions of NGF, BDNF and vascular endothelial growth factor in the injury field, promoting myelination, axonal growth and microvascular generation and nerve regeneration, muscle reinnervation and functional recovery. These results suggested that RCP/pDNA-PSPF@PGA, as an effective gene delivery platform, could provide a local gene therapy to improve nerve regeneration.

Enhanced nuclear gene delivery via integrating and streamlining intracellular pathway.

The essential challenge of gene therapy is to develop safe and efficient vectors that escort genes to target sites. However, due to the cumbersome workflow of gene transfection into cells, successive gene loss occurs. This leads to considerable reductions in nuclear gene uptake, eventually causing low gene expression. Herein, we designed a gene vector named CA3S2 (C: N,N'-cystamine-bis-acrylamide [CBA], A: agmatine dihydrochloride [Agm], S: 4-(2-aminoethyl) benzenesulfonamide [ABS]) with excellent gene transfection ability. This vector can promote gene delivery to the nucleus via enhanced endoplasmic reticulum (ER) targeting through integrating and streamlining of the complex intracellular pathway. Briefly, ABS endowed CA3S2/DNA nanoparticles with not only a natural ER-targeting tendency attributed to the caveolae-mediated pathway but also direct receptor-binding capacity on the ER surface. Agm enabled CA3S2 to enhance lysosomal escape and nuclear uptake ability. The gene delivery efficiency of CA3S2 was significantly better than that of polyethyleneimine 25K (PEI 25K). Therefore, CA3S2 is a promising gene carrier, and the ER-targeting strategy involving intracellular pathway integration and streamlining has potential for gene therapy.

Structured Liquid Droplets as Chemical Sensors that Function Inside Living Cells.

We report that micrometer-scale droplets of thermotropic liquid crystals (LCs) can be positioned inside living mammalian cells and deployed as chemical sensors to report the presence of toxins in extracellular environments. Our approach exploits droplets of LC enclosed in semi-permeable polymer capsules that enable internalization by cells. The LC droplets are stable in intracellular environments, but undergo optical changes upon exposure of cells to low, sub-lethal concentrations of toxic amphiphiles. Remarkably, LC droplets in intracellular environments respond to extracellular analytes that do not generate an LC response in the absence of cellular internalization. They also do not respond to other chemical stimuli or processes associated with cell growth or manipulation in culture. Our results suggest that droplet activation involves the transport and co-adsorption of amphiphilic toxins and other lipophilic cell components to the surfaces of internalized droplets. This work provides fundamentally new designs of biotic-abiotic systems that can report sensitively and selectively on the presence of select chemical agents outside cells and provides a foundation for the design of structured liquid droplets that can sense and report on other biochemical or metabolic processes inside cells.

Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6ß4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.

Hierarchical Self-assembly of Discrete Metal-Organic Cages into Supramolecular Nanoparticles for Intracellular Protein Delivery.

Hierarchical self-assembly (HAS) is a powerful approach to create supramolecular nanostructures for biomedical applications. This potency, however, is generally challenged by the difficulty of controlling the HAS of biomacromolecules and the functionality of resulted HAS nanostructures. Herein, we report a modular approach for controlling the HAS of discrete metal-organic cages (MOC) into supramolecular nanoparticles, and its potential for intracellular protein delivery and cell-fate specification. The hierarchical coordination-driven self-assembly of adamantane-functionalized M12 L24 MOC (Ada-MOC) and the host-guest interaction of Ada-MOC with ß-cyclodextrin-conjugated polyethylenimine (PEI-ßCD) afford supramolecular nanoparticles in a controllable manner. HAS maintains high efficiency and orthogonality in the presence of protein, enabling the encapsulation of protein into the nanoparticles for intracellular protein delivery for therapeutic application and CRISPR/Cas9 genome editing.

Enhanced antitumor effects of follicle-stimulating hormone receptor-mediated hexokinase-2 depletion on ovarian cancer mediated by a shift in glucose metabolism.

BACKGROUND: Most cancers favor glycolytic-based glucose metabolism. Hexokinase-2 (HK2), the first glycolytic rate-limiting enzyme, shows limited expression in normal adult tissues but is overexpressed in many tumor tissues, including ovarian cancer. HK2 has been shown to be correlated with the progression and chemoresistance of ovarian cancer and could be a therapeutic target. However, the systemic toxicity of HK2 inhibitors has limited their clinical use. Since follicle-stimulating hormone (FSH) receptor (FSHR) is overexpressed in ovarian cancer but not in nonovarian healthy tissues, we designed FSHR-mediated nanocarriers for HK2 shRNA delivery to increase tumor specificity and decrease toxicity. RESULTS: HK2 shRNA was encapsulated in a polyethylene glycol-polyethylenimine copolymer modified with the FSH ß 33-53 or retro-inverso FSH ß 33-53 peptide. The nanoparticle complex with FSH peptides modification effectively depleted HK2 expression and facilitated a shift towards oxidative glucose metabolism, with evidence of increased oxygen consumption rates, decreased extracellular acidification rates, and decreased extracellular lactate and glucose consumption in A2780 ovarian cancer cells and cisplatin-resistant A2780CP counterpart cells. Consequently, cell proliferation, invasion and migration were significantly inhibited, and tumor growth was suppressed even in cisplatin-resistant ovarian cancer. No obvious systemic toxicity was observed in mice. Moreover, the nanoparticle complex modified with retro-inverso FSH peptides exhibited the strongest antitumor effects and effectively improved cisplatin sensitivity by regulating cisplatin transport proteins and increasing apoptosis through the mitochondrial pathway. CONCLUSIONS: These results established HK2 as an effective therapeutic target even for cisplatin-resistant ovarian cancer and suggested a promising targeted therapeutic approach.

Bacterial magnetic particles-polyethylenimine vectors deliver target genes into multiple cell types with a high efficiency and low toxicity.

Bacterial magnetic particles (BMPs) are biosynthesized magnetic nano-scale materials with excellent dispersibility and biomembrane enclosure properties. In this study, we demonstrate that BMPs augment the ability of polyethylenimine (PEI) to deliver target DNA into difficult-to-transfect primary porcine liver cells, with transfection efficiency reaching over 30%. Compared with standard lipofection and polyfection, BMP-PEI gene vectors significantly enhanced the transfection efficiencies for the primary porcine liver cells and C2C12 mouse myoblast cell lines. To better understand the mechanism of magnetofection using BMP-PEI/DNA vectors, transmission electron microscopy (TEM) images of transfected Cos-7, HeLa, and HEP-G2 cells were observed. We found that the BMP-PEI/DNA complexes were trafficked into the cytoplasm and nucleus by way of vesicular transport and endocytosis. Our study builds support for the versatile BMP-PEI vector transfection system, which might be exploited to transfect a wide range of cell types or even to reach specific targets in the treatment of disease. KEY POINTS: • We constructed a BMP-PEI gene delivery vector by combining BMPs and PEI. • The vector significantly enhanced transfection efficiencies in eukaryotic cell lines. • The transfection mechanism of this vector was explained in our study.

Use of polyethylenimine to produce immobilized lipase multilayers biocatalysts with very high volumetric activity using octyl-agarose beads: Avoiding enzyme release during multilayer production.

A strategy to obtain biocatalysts formed by three enzyme layers has been designed using lipases A and B from Candida antarctica (CALA and CALB), the lipases from Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), and the artificial chimeric phospholipase Lecitase Ultra (LEU). The enzymes were initially immobilized via interfacial activation on octyl-agarose beads, treated with polyethylenimine (PEI) and a new enzyme layer was immobilized on the octyl-enzyme-PEI composite by ion exchange, producing octyl-enzyme-PEI-enzyme biocatalysts. Except when using LEU, when the two-layer biocatalysts, a large percentage of the PEI-immobilized enzyme was released when a new batch of PEI was added. This was prevented by glutaraldehyde crosslinking. The enzyme modifications produced more active preparations in some cases while in other cases, the effect of the modifications was negative for enzyme activity. These effects of the enzymes modifications were also different when the enzyme was immobilized by interfacial activation or by ion exchange. In all cases, the 3-layer biocatalysts were more active than the single- or bi-layer biocatalysts with some of the assayed substrates. However, as the substrate diffusion problems increased when new enzyme layers were added, even a decrease in enzyme activity with some substrates was found after increasing the number of enzyme layers.

Mesenchymal stem cells engineered by modified polyethylenimine polymer for targeted cancer gene therapy, in vitro and in vivo.

Cell-based delivery system is a promising strategy to protect therapeutic agents from the immune system and provide targeted delivery. Mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in cell-based gene therapy due to their unique features including tumor-tropic property and migratory ability. However, gene transfer into MSCs is limited due to low efficiency and cytotoxicity of carriers. In this study, we designed a novel delivery system based on polyethylenimine (PEI25 ) to improve these features of carrier and transfect plasmid encoding TRAIL to MSCs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand of TNF family with selective effect on cancerous cells. Then, death induction and migration ability of TRAIL-expressing MSCs was studied in melanoma cells. The effect of engineered-MSCs as an antitumor vehicle was also investigated in mice bearing melanoma cells. Our findings indicated that heterocyclic amine derivative of PEI25 showed significant improvement in MSCs viability determined by MTT assay and gene expression using fluorescent microscopy, flow cytometry, and Western blot analysis. We observed that engineered-MSCs could migrate toward and induce cell death in B16F0 cells in vitro. The single administration of TRAIL-expressing MSCs could delay tumor appearance and efficiently reduce tumor weights. Hematoxylin and eosin staining of tumor sections revealed extensive neoplastic cells necrosis. Furthermore, engineered-MSCs could migrate and localize to tumors sites within 5 days. Our results indicated that MSCs engineered by modified-PEI/TRAIL complexes could be considered as a promising cellular vehicle for targeted tumor suppression.

Virus-Inspired Mimics: Dual-pH-Responsive Modular Nanoplatforms for Programmable Gene Delivery without DNA Damage with the Assistance of Light.

Although tremendous efforts have been made to construct gene vectors incorporating multiple functionalities and moieties, designing gene vectors integrating innovative features to successfully negotiate biological impediments, which hamper efficacious responses in gene-based therapy, is still very urgent. Herein, a light-induced virus-inspired mimic, in which a modular envelope was utilized to mask polyethylenimine/DNA (PD) polyplexes, was developed based on two pH-responsive polymers. The virus-inspired envelope, which was capable of achieving multitargeting and dual-pH-responsiveness in endo/lysosomal compartments, was composed of an internalizing arginylglycylaspartic acid-modified module and a citraconic anhydride-modified nuclear localized signal-functionalized module. The envelope conjugated with chlorin e6 (Ce6) was shielded on the surface of PD polyplexes. Dual-pH-responsive deshielding of the virus-inspired mimic in endo/lysosomes allowed generation of a nonfatal amount of reactive oxygen species (ROS) under short-time photoirradiation, leading to photochemical internalization and much more substantial enhancement in light-induced gene expression without DNA damage caused by ROS. Confocal images revealed that the virus-inspired mimic achieved successful nuclear translocation of Ce6, resulting in nucleus-targeting photodynamic therapy (PDT). Furthermore, pTRAIL-mediated gene therapy, accompanied by a fatal amount of ROS under long-time photoirradiation, additionally consolidated in vitro antitumor outcomes. This study demonstrates a novel paradigm of "one arrow, two hawks," accomplishing a combination of enhanced gene therapy and PDT.

A programmable polymer library that enables the construction of stimuli-responsive nanocarriers containing logic gates.

Stimuli-responsive biomaterials that contain logic gates hold great potential for detecting and responding to pathological markers as part of clinical therapies. However, a major barrier is the lack of a generalized system that can be used to easily assemble different ligand-responsive units to form programmable nanodevices for advanced biocomputation. Here we develop a programmable polymer library by including responsive units in building blocks with similar structure and reactivity. Using these polymers, we have developed a series of smart nanocarriers with hierarchical structures containing logic gates linked to self-immolative motifs. Designed with disease biomarkers as inputs, our logic devices showed site-specific release of multiple therapeutics (including kinase inhibitors, drugs and short interfering RNA) in vitro and in vivo. We expect that this 'plug and play' platform will be expanded towards smart biomaterial engineering for therapeutic delivery, precision medicine, tissue engineering and stem cell therapy.

Surface chemistry governs the sub-organ transfer, clearance and toxicity of functional gold nanoparticles in the liver and kidney.

BACKGROUND: To effectively applied nanomaterials (NMs) in medicine, one of the top priorities is to address a better understanding of the possible sub-organ transfer, clearance routes, and potential toxicity of the NMs in the liver and kidney. RESULTS: Here we explored how the surface chemistry of polyethylene glycol (PEG), chitosan (CS), and polyethylenimine (PEI) capped gold nanoparticles (GNPs) governs their sub-organ biodistribution, transfer, and clearance profiles in the liver and kidney after intravenous injection in mice. The PEG-GNPs maintained dispersion properties in vivo, facilitating passage through the liver sinusoidal endothelium and Disse space, and were captured by hepatocytes and eliminated via the hepatobiliary route. While, the agglomeration/aggregation of CS-GNPs and PEI-GNPs in hepatic Kupffer and endothelial cells led to their long-term accumulation, impeding their elimination. The gene microarray analysis shows that the accumulation of CS-GNPs and PEI-GNPs in the liver induced obvious down-regulation of Cyp4a or Cyp2b related genes, suggesting CS-GNP and PEI-GNP treatment impacted metabolic processes, while the PEI-GNP treatment is related with immune responses. CONCLUSIONS: This study demonstrates that manipulation of nanoparticle surface chemistry can help NPs selectively access distinct cell types and elimination pathways, which help to clinical potential of non-biodegradable NPs.

PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics.

Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1 mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.

Enabling Combinatorial siRNA Delivery against Apoptosis-Related Proteins with Linoleic Acid and α-Linoleic Acid Substituted Low Molecular Weight Polyethylenimines.

PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.