Preparation of Effective and Safe Gene Carriers by Grafting Alkyl Chains to Generation 5 Polypropyleneimine.

Gene therapy is a novel method to treat a variety of diseases including genetic disorders and cancer. Nonviral gene carriers have now gained considerable attention as gene carrier systems. Polyamidoamine (PAMAM) and polypropyleneimine (PPI) are the two most widely used denderimers in gene delivery studies. The aim of the current study was to investigate the effects of modification of generation 5 polypropyleneimine (G5 PPI) dendrimers with alkanoate groups as hydrophobic moieties on DNA transfection and cytotoxicity. Six, 10, and 16 carbon derivatives of bromoalkanoic acids were conjugated onto PPI with 10%, 30%, and 50% of surface amine grafting. Ethidium bromide exclusion assay results proved the ability of modified carriers to condense DNA. Transfection assay showed higher DNA delivery potential for 30% and 50% grafting with decanoate moieties compared to native G5 PPI and Superfect(TM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) and apoptosis experiments showed lower toxicity for modified carriers compared to unmodified PPI. The hemolytic effect of grafted carriers was not significantly different from G5 PPI. Size and zeta potential measurements revealed that polyplex size was less than 200 nm and electrical charges were in the range 14-25 mV. The hydrophobic modifications improved transfection activity and toxicity of G5 PPI without negatively affecting hemocompatibility. These modified carriers are therefore promising candidates for further in vivo investigations.

The potential of mesenchymal stem cell coexpressing cytosine deaminase and secretory IL18-FC chimeric cytokine in suppressing glioblastoma recurrence.

Glioblastoma multiforme (GBM) patients have a high recurrence rate of 90%, and the 5-year survival rate is only about 5%. Cytosine deaminase (CDA)/5-fluorocytosine (5-FC) gene therapy is a promising glioma treatment as 5-FC can cross the blood-brain barrier (BBB), while 5-fluorouracil (5-FU) cannot. Furthermore, 5-FU can assist reversing the immunological status of cold solid tumors. This study developed mesenchymal stem cells (MSCs) co-expressing yeast CDA and the secretory IL18-FC superkine to prevent recurrent tumor progression by simultaneously exerting cytotoxic effects and enhancing immune responses. IL18 was fused with Igk and IgG2a FC domains to enhance its secretion and serum half-life. The study confirmed the expression and activity of the CDA enzyme, as well as the expression, secretion, and activity of secretory IL18 and IL18-FC superkine, which were expressed by lentiviruses transduced-MSCs. In the transwell tumor-tropism assay, it was observed that the genetically modified MSCs retained their selective tumor-tropism ability following transduction. CDA-expressing MSCs, in the presence of 5-FC (200 µg/ml), induced cell cycle arrest and apoptosis in glioma cells through bystander effects in an indirect transwell co-culture system. They reduced the viability of the direct co-culture system when they constituted only 12.5 % of the cell population. The effectiveness of engineered MSCs in suppressing tumor progression was assessed by intracerebral administration of a lethal dose of GL261 cells combined in a ratio of 1:1 with MSCs expressing CDA, or CDA and sIL18, or CDA and sIL18-FC, into C57BL/6 mice. PET scan showed no conspicuous tumor mass in the MSC-CDA-sIL18-FC group that received 5-FC treatment. The pathological analysis showed that tumor progression suppressed in this group until 20th day after cell inoculation. Cytokine assessment showed that both interferon-gamma (IFN-γ) and interleukin-4 (IL-4) increased in the serum of MSC-CDA-sIL18 and MSC-CDA-sIL18-FC, treated with normal saline (NS) compared to those of the control group. The MSC-CDA-sIL18-FC group that received 5-FC treatment showed reduced serum levels of IL-6 and a considerably improved survival rate compared to the control group. Therefore, MSCs co-expressing yeast CDA and secretory IL18-FC, with tumor tropism capability, may serve as a supplementary approach to standard GBM treatment to effectively inhibit tumor progression and prevent recurrence.

Targeted doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting against colorectal cancer.

Exosomes hold great potential for cancer treatment to deliver therapeutics due to its inherent low immunogenicity. Exosomes are biocompatible cell-exocytosed secreted vesicles by most cell types, which can be used to construct novel biomanufacturing platform for drug delivery and cancer therapy. In this study, we implemented nano-sized vesicles which were secreted by mesenchymal stem cell (MSC), to encapsulate doxorubicin (DOX) through electroporation method (DOX@exosome). DOX was loaded into exosomes, with an encapsulation efficiency of up to 35% and separated by ultracentrifugation. Subsequently, carboxylic acid-end MUC1 aptamer was used to covalently decorate the surface amine groups of the exosomes via amide bond formation to provide selective guided drug delivery (DOX@exosome-apt). The data showed that the DOX@exosome-apt provided highly efficient DOX transportation to MUC1-positive cancer cells in vitro as confirmed by MTT and flow cytometry experiments. Moreover, in vivo study on ectopic model of C26 (mouse colon adenocarcinoma) in BALB/c mice indicated that the single dose intravenous injection of DOX@exosome-apt significantly suppress tumor growth in comparison with free DOX. Ex vivo fluorescent imaging also verified the desirable biodistribution of DOX@exosome-apt by exhibiting higher tumor accumulation and faster liver clearance in comparison with DOX@exosome and free DOX. It could be concluded that MUC1 aptamer-decorated exosomes can be implemented therapeutically for the safe and versatile delivery of DOX to colon adenocarcinoma, thus offering valuable platform for clinical applications.

Effective and safe in vivo gene delivery based on polyglutamic acid complexes with heterocyclic amine modified-polyethylenimine.

Polyethylenimine (PEI) has been extensively used for non-viral gene delivery. Increasing the molecular weight of PEI often improves transfection efficiency, but enhances cytotoxicity and non-specific interaction with plasma proteins, limiting its use in clinical applications. In this study, poly-l-glutamic acid (L-PGA) as an anionic polymer, was introduced to piperazine-modified PEI to improve its in vivo properties. The physicochemical properties, cytotoxicity, in vitro and in vivo tranfection efficiency of these carriers were evaluated. Conjugation of 50% of primary amines of PEI 25 kDa with piperazine in the presence of PGA1% (PEI25Pip50%/PGA1%) could significantly increase transfection efficiency even in the presence of serum compared to PEI 25 kDa. Increasing the PGA content led to lower cytotoxicity of DNA/PEI25Pip50%/PGA1% triplexes. Systemic administration of triplexes in Balb/c mice resulted in significant enhancement of luciferase gene expression in brain, spleen, and liver compared to PEI 25 kDa. In a 30-day survival study, no significant changes were observed in mice body weights in DNA/PEI25Pip50%/PGA1% group. Moreover, this group exhibited a survival rate of 100% compared to 0% in mice receiving PEI 25 kDa. This novel PEI25Pip50%/PGA1% carrier could be used to overcome the serum inhibitory effects on gene expression in vivo, providing a promising gene delivery system for tissue-specific targeting.

Smart AS1411-aptamer conjugated pegylated PAMAM dendrimer for the superior delivery of camptothecin to colon adenocarcinoma in vitro and in vivo.

In the current study camptothecin-loaded pegylated PAMAM dendrimer were synthesized and were functionalized with AS1411 anti-nucleolin aptamers for site-specific targeting against colorectal cancer cells which over expresses nucleolin receptors. The morphological properties and size dispersity of the prepared nanoparticles were evaluated using transmission electron microscope (TEM) and DLS. The drug-loading content and encapsulation efficiency were obtained 8.1% and 93.67% respectively. The in vitro release of camptothecin from the formulation was provided the sustained release of encapsulated camptothecin during 4days. Comparative in vitro cytotoxicity experiments demonstrated that the targeted camptothecin loaded-pegylated dendrimers had higher antiproliferation activity, towards nucleolin-positive HT29 and C26 colorectal cancer cells than nucleolin-negative CHO cell line. Fluorscence microscopy and flow cytometry also confirmed the enhanced cellular uptake of AS1411 targeted pegylated-dendrimer. In vivo study in C26 tumor-bearing BALB/C mice revealed that the AS1411-functionalized camptothecin loaded pegylated dendrimers improved antitumor activity and survival rate of the encapsulated camptothecin. Conjugation of AS1411 aptamer to the camptothecin loaded-pegylated dendrimer surface provides site-specific delivery of camptothecin, inhibit C26 tumor growth in vivo and significantly decrease systemic toxicity. These results suggested that the new nucleolin-targeted pegylated PAMAM dendrimer as a delivery system for camptothecin have the potential for the treatment of nucleolin-overexpressed colorectal cancer.

PAMAM-pullulan conjugates as targeted gene carriers for liver cell.

Targeted nano-carriers are highly needed to promote nucleic acid delivery into the specific cell for therapeutic approaches. Pullulan as a linear carbohydrate has an intrinsic liver targeting property interacting with asialoglycoprotein receptor (ASGPR) found on liver cells. In the present study, we developed polyamidoamine (PAMAM)-pullulan conjugates and investigated their targeting activity in delivering gene into liver cells. The particle size, zeta potential, buffering capacity and ethidium bromide exclusion assays of the conjugates were evaluated. The cytotoxicity and transfection efficiency of new derivatives were assessed following in vitro transfection of HepG2 (receptor positive) and N2A (receptor negative) cell lines. Size of conjugated polymers ranged between 118 and 184 nanometers and their cytotoxicity were similar to PAMAM. Among six produced nanocarriers, G4PU4 and G5PU4 enhanced transfection efficiency in HepG2 cells compared to unmodified PAMAM. Therefore, the PAMAM-pullulan derivatives seem to improve delivery of nucleic acids into the liver cells expressing asialoglycoprotein receptor with minimal transfection in non-targeted cells.

Tetrac-conjugated polymersomes for integrin-targeted delivery of camptothecin to colon adenocarcinoma in vitro and in vivo.

In this study, we prepared tetraiodothyroacetic acid (tetrac) conjugated PEG-PLGA polymersomes for the targeted delivery of camptothecin to colon adenocarcinoma. Tetrac, which binds to integrin αvß3 with high affinity and specificity, was covalently conjugated to the surface of the PEGylated polymersomal formulation of camptothecin (CPT). The hydrodynamic and morphological properties of the prepared system were evaluated using TEM (transmission electron microscopy), SEM (scanning electron microscopy) and DLS (dynamic light scattering) experiments. Camptothecin was encapsulated in the polymersomal system with encapsulation efficiency and loading content of 84±10.12 and 4.2±0.82, respectively. The in vitro release profile of camptothecin from the polymersomal formulation revealed the sustained release pattern. In vitro cytotoxicity experiments confirmed that the tetrac-conjugated camptothecin loaded-polymersomes had higher cellular toxicity towards integrin-overexpressed HT29 and C26 colorectal cancer cells than integrin-negative CHO cell line. The in vivo tumor inhibitory effect of tetrac-conjugated camptothecin loaded-polymersomes demonstrated an enhanced therapeutic index of integrin targeted polymersomal formulation over both non-targeted polymersomal formulation and free camptothecin in C26 tumor bearing mice. The obtained results demonstrated that the prepared tetrac-conjugated polymersomes were able to control the release of camptothecin, and significantly increase the therapeutic index of compthotecin. This study demonstrates the versatility of integrin-targeted tetrac-conjugated PEG-PLGA polymersomal formulation as an anti-cancer nano-pharmaceutical platform.

Alkyl cross-linked low molecular weight polypropyleneimine dendrimers as efficient gene delivery vectors.

OBJECTIVES: In recent years, polypropyleneimine (PPI) dendrimers have attracted great interest as non-viral gene delivery systems because of their attractive features including highly branched architecture with number of reactive end groups. However, without being structurally modified, they are not efficient gene carriers. In the present study, generation 2 and 3 (G2 and G3) of PPI dendrimers were conjugated with alkylcarboxylate groups as linker to enhance the transfection efficiency while maintaining their low cell toxicity. MATERIALS AND METHODS: First, 10-bromodecanoic acid was covalently attached to all available surface primary amines of PPI G2 and G3 to increase their lipophilicity. In the subsequent step, PPIs were conjugated to the alkylcarboxylate groups of alkylcarboxylate-PPI derivatives to increase the number of surface primary amines. Physicochemical properties of modified PPIs were determined. Transfection experiments (using both luciferase and green fluorescent protein (GFP)- expressing plasmids) and cytotoxicity assay were performed to evaluate the efficiency of the final derivatives. RESULTS: Fabricated vectors condensed DNA effectively so that polyplexes with appropriate size (below 155 nm) and positive surface charge were constructed. Cross-linked low molecular weight PPIs (G2 or G3) with decanoate linkage increased transfection efficiency significantly while maintaining the low cytotoxicity. PPI G2 derivative exhibited increased buffering capacity which is believed to be responsible for better proton sponge mechanism leading to higher transfection efficiency. CONCLUSION: Our results indicated that oligomerization of low molecular weight PPI (PPI G2-alkyl-PPI G2 conjugate) could be an approach to increase the transfection efficiency and to lower the cytotoxicity of low molecular weight polycations.

Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors.

One strategy for improving gene vector properties of polyethylenimine is to facilitate individual transfection mechanism steps. This study investigates (i) improving transfection efficiency by attaching peptide nuclear localization signals (nuclear localization signals: SV40 large T antigen nuclear localization signal or C-terminus of histone H1) to polyethylenimine (10 kDa) and (ii) using disulfide linkages, which are expected to be stable during polyplex formation, but cleaved inside cells giving improved gene release. Nuclear localization signal-containing polyplexes exhibited low cytotoxicity, whereas transfection efficiency with high molecular weight plasmid DNA increased up to 3.6 times that of underivatized polyethylenimine in Neuro2A cells at higher molar ratio of polyethylenimine-nitrogen to DNA-phosphate (N/P) ratios. However, with luciferase-specific low molecular weight small interfering RNA in Neuro2A/EGFPLuc cells, nuclear localization signal-containing polyplexes with disulfide linkages caused substantial cytotoxicity at N/P ratios >15 and no consistent significant reduction in luciferase expression. Possible explanations for molecular weight-dependent differences in genetic information transfer by polyplexes containing disulfide-linked nuclear localization signals are discussed.