Fabrication of anionic dextran-coated micelles for aptamer targeted delivery of camptothecin and survivin-shRNA to colon adenocarcinoma.

In this study, we synthesized PLA-PEI micelles which was co-loaded with an anticancer drug, camptothecin (CPT), and survivin-shRNA (sur-shRNA). The hydrophobic CPT was encapsulated in the core of the polymeric micelles while sur-shRNA was adsorbed on the shell of the cationic micelles. Then, the positively-charged sur-shRNA-loaded micelles were coated with poly carboxylic acid dextran (PCAD) to form PLA/PEI-CPT-SUR-DEX. To selectively target the system to colon cancer cells, AS1411 aptamer was covalently attached to the surface of the PCAD-coated nanoparticles (PLA/PEI-CPT-SUR-DEX-APT). PLA/PEI-CPT-SUR-DEX-APT enhanced cellular uptake through receptor-mediated endocytosis followed by increased CPT accumulation, downregulation of survivin, and thereby 38% cell apoptosis. In C26 tumor-bearing mice models, after administered intravenously, PLA/PEI-CPT-SUR-DEX-APT and PLA/PEI-CPT-SUR-DEX formulations resulted in a significant inhibition of the tumor growth with tumor inhibition rate of 93% and 87%, respectively. Therefore, PLA/PEI-CPT-SUR-DEX-APT could be a versatile co-delivery vehicle for promising therapy of colorectal cancer.

Synthesis of chimeric polymersomes based on PLA-b-PHPMA and PCL-b-PHPMA for nucleoline guided delivery of SN38.

We reported SN38-loaded polymersomes formulated with amphiphilic block copolymers based on HPMA and either ε-caprolactone or lactic acid through employing ring-opening polymerization, carbodiimide chemistry and a reversible addition-fragmentation chain transfer polymerization technique. In this regard, we successfully synthesized five chimeric polymersomes based on different percentage of the synthesized copolymers. The prepared chimeric polymersomes based on PCL-b-PHPMA:PLA-b-PHPMA at ratio of 1:3 exhibited superior loading capacity in comparison with other chimeric polymersomes. In order to increase therapeutic index of the prepared systems, AS1411 aptamer was implemented as targeting ligand. In vivo study revealed that the intravenous single dose injection of targeted chimeric polymersomes to C26 tumor bearing mice had remarkable efficacy in inhibiting tumor growth. It could be concluded that the chimeric polymersomes fabricated from PCL-b-PHPMA and PLA-b-PHPMA at a ratio of 1:3 have great potential for SN38 encapsulation while providing controlled sustained release properties with targeting capability via AS1411 aptamer conjugation.

Evaluation of Efficiency of Modified Polypropylenimine (PPI) with Alkyl Chains as Non-viral Vectors Used in Co-delivery of Doxorubicin and TRAIL Plasmid.

In this study, co-delivery system was achieved via plasmid encoding TNF related apoptosis inducing ligand (pTRAIL) and doxorubicin (DOX) using carrier based on polypropylenimine (PPI) modified with 10-bromodecanoic acid. Incorporation of alkylcarboxylate chain to PPIs (G4 and G5) could improve transfection efficiency via overcoming the plasma membrane barrier of the cells and decrease cytotoxicity of PPI. Characterization of fabricated NPs revealed that PPI G5 in which 30% of primary amines were substituted by alkyl carboxylate chain (PPI G5-Alkyl 30%) has higher drug loading as compared to the other formulations. PPI G5-Alkyl 30% indicated a decreased drug release may be due to alkyl chains on the surface of PPI, which serve as an additional hindrance for drug diffusion. In vitro cytotoxicity experiments demonstrated that co-delivery system induced apoptosis of tumor cells more efficiently than each of delivery system alone. Furthermore, these results revealed that our combined delivery platform of pTRAIL and DOX using Alkyl-modified PPI G5 can significantly improve the anti-tumor activity and this strategy might develop a new therapeutic window for cancer treatment.

Surface engineering of hollow gold nanoparticle with mesenchymal stem cell membrane and MUC-1 aptamer for targeted theranostic application against metastatic breast cancer.

Mesenchymal stem cell membrane (MSCM)-coated biomimetic doxorubicin-loaded hollow gold nanoparticles were fabricated and decorated with MUC1 aptamer in order to provide smart theranostic platform. The prepared targeted nanoscale biomimetic platform was extensively characterized and evaluated in terms of selective delivery of DOX and CT-scan imaging. The fabricated system illustrated spherical morphology with 118 nm in diameter. Doxorubicin was loaded into the hollow gold nanoparticles through physical absorption technique with encapsulation efficiency and loading content of 77%±10 and 31%±4, respectively. The in vitro release profile demonstrated that the designed platform could respond to acidic environment, pH 5.5 and release 50% of the encapsulated doxorubicin during 48 h, while 14% of the encapsulated doxorubicin was released in physiological condition, pH 7.4 up to 48 h. The in vitro cytotoxicity experiments on 4T1 as MUC1 positive cell line illustrated that the targeted formulation could significantly increase mortality at 0.468 and 0.23 µg/ml of equivalent DOX concentration compared to non-targeted formulation while this cytotoxicity was not observed in CHO as MUC1 negative cell line. Furthermore, in vivo experiments showed high tumor accumulation of the targeted formulation even 24 h after intravenous injection which induced effective tumor growth suppression against 4T1 tumor bearing mice. On the other hand, existence of hollow gold in this platform provided CT scan imaging capability of the tumor tissue in 4T1 tumor bearing mice up to 24 h post-administration. The obtained results indicated that the designed paradigm are promising and safe theranostic system for fighting against metastatic breast cancer.

Theranostic nanobubbles towards smart nanomedicines.

Targeted therapy and early accurate detection of malignant lesions are essential for the effectiveness of treatment and prognosis in cancer patients. The development of gaseous system as a versatile platform for the fabricated nanobubbles, has attracted much interest in improving the efficacy of ultrasound therapeutic, diagnostic, and theranostic platforms. Nano-sized bubble, as an ultrasound contrast agent, with spherical gas-filled structures exhibited contrast enhancement capability due to their inherent EPR effect. Additionally, nanobubbles exhibited good stability with extended retention time in the blood stream. The current review summarized various nanobubbles and discussed about the crucial parameters affecting the stability of ultrafine bubbles. Furthermore, therapeutic and theranostic gaseous systems for fighting against cancer were described.

Hybrid carbon-based materials for gene delivery in cancer therapy.

Accumulation at tumor tissue without any damage to healthy normal tissues is an ultimate goal in cancer therapy. Despite many efforts in the field of cancer therapy, this disease remains as the major reason of mortality all over the world. Gene therapy has introduced great opportunity to fight against cancer disease. It should be noted that still some obstacles limit clinical application of gene delivery approach, which have to be overcome for efficient transportation of therapeutic gene to the site of action. In this regard, carbon nanomaterials and their unique physical and chemical properties such as their capability of DNA protection have attracted much attention in the field of nanomedicine and non-viral carriers for therapeutic genes. Although, negligible solubility of carbon nanomaterials in biological environments has limited their biomedical application but their structural characteristics facilitate their chemical modifications thereby overcoming their solubility problem. Moreover, hybridization of modified carbon materials with different polymers provides more biocompatible and capable systems for gene delivery purposes. In the current review, we summarized hybrid carbon-based materials as non-viral carriers for gene delivery.

Targeted rod-shaped mesoporous silica nanoparticles for the co-delivery of camptothecin and survivin shRNA in to colon adenocarcinoma in vitro and in vivo.

Simultaneous drug and gene delivery to cancer cells has been introduced to provide advantages of the synergistic effects of gene to sensitize the cancer cells to chemotherapeutic agent. In the current study, nucleolin-targeted co-delivery system, based on PEGylated rod-shaped mesoporous silica NPs was developed as a biocompatible nanocarrier for simultaneous delivery of camptothecin and survivin shRNA-expressing plasmid (iSur-DNA) to colon adenocarcinoma. The structural characterization including hydrodynamic radius and morphological characteristics of the prepared system demonstrated the mesoporous rod-shaped structure of the prepared system with 100-150 nm diameter. Camptothecin was loaded into the rod-shaped MSN NPs with encapsulation efficiency of 32%. At the next stage, the prepared camptothecin-loaded system was PEGylated and then iSur-DNA was condensed with C/P ratio of 6 to form PEG@MSNR-CPT/Sur. Then, the prepared camptothecin-iSur-DNA loaded PEGylated rod-shaped mesoporous silica NPs were tagged with AS1411 DNA aptamer (Apt-PEG@MSNR-CPT/Sur) in order to provide selective therapy against colorectal adenocarcinoma. The obtained results showed that the prepared platform controlled the release of anticancer drug, camptothecin. The experimental results indicated potent synergistic effect of iSur-pDNA and CPT in in vitro cytotoxicity, apoptosis induction and in vivo antitumor effect. In addition, tagging the system with AS1411 DNA aptamer facilitated drug uptake into nucleolin positive colorectal cancer cells leading to higher cellular toxicity and apoptosis induction in C26 cells compared to nucleolin-negative CHO cell line. Apt-PEG@MSNR-CPT/Sur system significantly supressed tumor growth rate in C26 tumor bearing mice while improving survival rate and pharmacokinetics of the platform in comparison with PEG@MSNR-CPT and PEG@MSNR-CPT/Sur. It could be concluded that the developed nucelolin targeted nanomedicine for co-delivery of camptothecin and iSur-DNA could serve as a versatile nanotherapeutic system against colorectal cancer.

Polyethylenimine-functionalized carbon nanotubes tagged with AS1411 aptamer for combination gene and drug delivery into human gastric cancer cells.

In this project, synergistic cancer cell death was achieved by a targeted delivery system comprising Bcl-xL-specific shRNA and a very low DOX content, which simultaneously activated an intrinsic apoptotic pathway. A modified branched polyethylenimine (PEI 10kDa) was grafted through polyethylene glycol (PEG) linker to carboxylated single-walled carbon nanotubes (SWCNT) to serve as a vehicle for shRNA delivery. The SWNT-PEG-PEI conjugate was covalently attached to AS1411 aptamer as the nucleolin ligand to target the co-delivery system to the tumor cells overexpressing nucleolin receptors on their surface. The final vehicle was eventually obtained after intercalation of DOX with pBcl-xL shRNA-SWCNT-PEG-10-10%PEI-Apt. Cell viability assay, GFP expression and transfection experiment against L929 (-nucleolin) and AGS (+nucleolin) cells illustrated that the tested targeted delivery system inhibited the growth of nucleolin-abundant gastric cancer cells with strong cell selectivity. Subsequently, we illustrated that the combination treatment of the selected shRNAs and DOX had excellent tumoricidal efficacy as verified by MTT assay. Furthermore, very low concentration of DOX, approximately 58-fold lower than its IC50 concentration, was used which could mitigate toxic side effects of DOX. Overall, our work revealed that combination of shRNA-mediated gene-silencing strategy with chemotherapeutic agents constitutes a valuable and safe approach for antitumor activity.

Co-delivery of Doxorubicin Encapsulated PLGA Nanoparticles and Bcl-xL shRNA Using Alkyl-Modified PEI into Breast Cancer Cells.

In recent years, much effort has been focused on an appropriate combination of chemotherapeutic drugs and nucleic acids to exploit additive or synergistic therapeutic effects and overcome many obstacles such as the reduction of side effects and drug resistance. Short hairpin RNA (shRNA) has designed to allow the production of small interfering RNA (siRNA) within the cells and offer long-lasting silencing of target genes. In this study, alkyl-modified polyethylenimine (PEI 10 kD) was used for co-delivery of doxorubicin (DOX) encapsulated into poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and Bcl-xL shRNA (one class of molecules that block apoptosis of tumor cells) into breast cancer cells. Our results demonstrated that modification of PEI with alkyl chain could enhance the induction of apoptosis in tumor cells by suppression of Bcl-xL gene using Bcl-xL shRNA more than PEI alone. On the other hand, DOX encapsulated into PLGA had more synergistic effect with shRNA in comparison with DOX alone. In conclusion, combination of PLGA-DOX NPs and alkyl-PEI/shRNA complexes may have promising applications in breast cancer therapy.

Chitosan-modified PLGA nanoparticles tagged with 5TR1 aptamer for in vivo tumor-targeted drug delivery.

In this study, we reported epirubicin (Epi) encapsulated nanoparticles (NPs) formulated with biocompatible and biodegradable poly (lactic-co-glycolic acid) (PLGA) modified with chitosan (CS) through a physical adsorption method. Using chitosan, the solubility and surface charge of PLGA was modified to make efficient drug carriers for cancer cells. To improve the anti-tumor efficacy, we developed targeted therapy of tumor cells using a 5TR1 DNA aptamer (Apt) against the MUC1 receptor. To prove the MUC1 receptor-mediated uptake of Epi-PLGA-CS-Apt NPs in the cells, competition experiments were carried out. In vitro experiments, cytotoxicity assay and fluorescence uptake assay demonstrated that fabricated NPs with or without aptamers showed significantly high therapeutic efficiency in MCF7 cells (breast cancer cell) compared with free Epi, while in BALB/c mice bearing C26 cells (murine colon carcinoma cell), targeted NP groups exhibited significant tumor growth inhibition and higher inclination to tumor compared with non-targeted NPs. Hence, our in vivo results revealed that non-targeted NPs may diffuse away from the tumor site and release Epi in the extracellular space and decrease concentration of the drug in the targeted tissue. This study indicated Epi-PLGA-CS-Apt has great potential as a promising nanoplatform for in vivo cancer therapy and could be of great value in medical use.

Extensive preclinical investigation of polymersomal formulation of doxorubicin versus Doxil-mimic formulation.

Due to the severe cardiotoxicity of doxorubicin, its usage is limited. This shortcoming could be overcome by modifying pharmacokinetics of the drugs via preparation of various nanoplatforms. Doxil, a well-known FDA-approved nanoplatform of doxorubicin as antineoplastic agent, is frequently used in clinics in order to reduce cardiotoxicity of doxorubicin. Since Doxil shows some shortcomings in clinics including hand and food syndrome and very slow release pattern thus, there is a demand for the development and preparation of new doxorubicin nanoformulation with fewer side effects. The new formulation of the doxorubicin, synthesized previously by our group was extensively examined in the current study. This new formulation is doxorubicin encapsulated in PEG-PLGA polymersomes (PolyDOX). The main aim of the study was to compare the distribution and treatment efficacy of a new doxorubicin-polymersomal formulation (PolyDOX) with regular liposomal formulation (Doxil-mimic) in murine colon adenocarcinoma model. Additionally, the pathological, hematological changes, pharmacodynamics, biodistribution, tolerated dose and survival rate in vivo were evaluated and compared. Murine colon cancer model was induced by subcutaneous inoculation of BALB/c mice with C26 cells. Afterwards, either Doxil-mimic or PolyDOX was administered intravenously. The obtained results from biodistribution study showed a remarkable difference in the distribution of drugs in murine organs. In this regard, Doxil-mimic exhibited prolonged (48h) presence within liver tissues while PolyDOX preferentially accumulate in tumor and the presence in liver 48h post-treatment was significantly lower than that of Doxil-mimic. Obtained results demonstrated comparable final length of life for mice receiving either Doxil-mimic or PolyDOX formulations whereas tolerated dose of mice receiving Doxil-mimic was remarkably higher than those receiving PolyDOX. Therapeutic efficacy of formulation in term of tumor growth rate after one injection of formulations (5mg/kg, 10mg/kg or 15mg/kg) demonstrated better efficacy at lower dose for PolyDOX. Analysis of Kaplan Meier curve was in favor of both formulations in their treatment-dose. Pathological and hematological surveys of mice treated with both formulations did not show considerable difference except for a small atrophy in liver observed after successive administration of Doxil-mimic. It could be concluded that PolyDOX can potentially limit off-site effects of Doxil due to its biodegradability and sustained release properties while it exhibited favorable safety profile comparable to Doxil.

High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region.

For high-throughput production of recombinant protein in Escherichia coli (E. coli), besides important parameters such as efficient vector with strong promoter and compatible host, other important issues including codon usage, rare codons, and GC content specially at N-terminal region should be considered. In the current study, the effect of decreasing the percentage of GC nucleotides and optimizing codon usage at N-terminal region of human growth hormone (hGH) cDNA on the level of its expression in E. coli were investigated. Mutation in cDNA of hGH was performed through site-directed mutagenesis using PCR. Then, the mutant genes were amplified and cloned into the expression vector, pET-28a. The new constructs were transformed into the BL21(DE3) strain of E. coli and chemically induced for hGH expression. At the final stage, expressed proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), scanning gel densitometry, and western blot. SDS-PAGE scanning gel densitometry assay and western blot analysis revealed higher expression level of hGH by using the two new expressions constructs (mutant genes vectors with decreasing GC content and optimized-codon usage at N-terminal of cDNA) in comparison with wild gene expression vector. Obtained results demonstrated that decreasing the GC nucleotide content and optimization of codon usage at N-terminal of the hGH cDNA could significantly enhance the expression of the target protein in E. coli. Our results highlight the important role of both 5´ region of the heterologous genes in terms of codon usage and also GC content on non-host protein expression in E. coli.

Evaluation of chemical modification effects on DNA plasmid transfection efficiency of single-walled carbon nanotube-succinate- polyethylenimine conjugates as non-viral gene carriers.

Polyethylenimine (PEI) is a widely used non-viral vector for DNA delivery. One major obstacle of higher molecular weight PEIs is the increased cytotoxicity despite the improved transfection efficiency and numerous chemical modifications that have been reported to overcome this problem. Carbon nanotubes (CNT) are carbon nanomaterials capable of penetrating into cell membranes with no cytotoxic effects. Covalent and noncovalent functionalization methods have been used to improve their solubility in aqueous media. The idea of conjugating PEIs and CNT through different chemical bonds and linkers seems promising as it may result in highly effective carriers due to combination of the transfection ability of PEI with cell internalization of CNT. In this study, six different water-soluble PEI conjugates of single-walled carbon nanotubes (SWNTs) were prepared by grafting PEI with one of three molecular weights (1.8, 10 and 25 kDa) through succinate as a linker which refers to "an organic moiety through which a SWNT is conjugated to PEI." The succinate linker was introduced to the surface of SWNTs through two different chemical strategies: a) ester and b) acyl linkages. The resulting SWNT-PEI vectors were characterized by IR spectroscopy, thermogravimetric analysis (TGA) and SEM imaging. All synthesized carriers were evaluated and compared for their cytotoxicity and transfection efficiency in murine neuroblastoma cells as polyplexes with plasmid DNA for luciferase and green fluorescent protein (GFP). The most efficient carriers were prepared by attaching PEI with the lowest molecular weight (1.8 kDa) through acyl linkage, which gave a transfection efficiency 190-fold greater than that of the corresponding free PEI. Transfection efficiency was the highest in polyplexes prepared with acyl-linked conjugates in all the plasmid/vector ratios studied.

Preparation and evaluation of polyethylenimine-functionalized carbon nanotubes tagged with 5TR1 aptamer for targeted delivery of Bcl-xL shRNA into breast cancer cells.

In this study, single-walled carbon nanotubes (SWCNTs) were covalently attached to poly(ethylene glycol) (PEG) and polyethylenimine (PEI) 10 kDa, or its derivatives, to fabricate efficient carriers for gene delivery. PEI 10 kDa was modified by alkylcarboxylation of its primary amines with a series of ω-bromo-alkylcarboxylic acids to provide a range of vectors with increased lipophilicity. PEI 10 kDa or its alkylcarboxylate derivatives were conjugated to SWCNT-PEG to develop vectors possessing effective DNA condensation ability which can interact with cell membrane via both nano-needle mechanism and electrostatic interactions produced by SWCNT and PEI, respectively. The results demonstrated that SWCNT-PEG-PEI and SWCNT-PEG-derivatives of PEI could condense DNA into particle size less than 150 nm with positive surface charges between 6.3-30.8 mV. To improve the antitumor efficacy, we developed a targeted gene delivery system using a 5 TR1 aptamer. The most efficient vector, which was prepared by attachment of SWCNT-PEG to modified PEI 10 kDa with 10-bromodecanoic acid (10%), showed 8.5-10 folds enhancement in transfection activity at C/P ratio 6 as compared to the gold standard PEI 25 kDa at C/P ratio of 0.8. We also showed that the selected polyplex could efficiently and selectively transfer plasmid shRNA to MUC1 positive cells.

A facile Friedel-Crafts acylation for the synthesis of polyethylenimine-grafted multi-walled carbon nanotubes as efficient gene delivery vectors.

Low chemical reactivity of carbon nanotubes is one of the major obstacles in their functionalization via chemical reactions. As a non-destructive method, Friedel-Crafts acylation was suggested among the explored reactions for which only a few methods have been reported under harsh reaction conditions, e.g., high temperature all leading to low yields. In this study, we propose a novel method for the acylation of multi-walled carbon nanotubes (MWCNTs) at a low temperature (i.e., 42°C), using SiO2-Al2O3 as a catalyst and 6-bromohexanoic acid as the acylating agent to produce high yield functionalized MWCNTs. After acylation, MWCNTs are conjugated with polyethylenimines (PEIs) with three molecular weights (1.8, 10 and 25kDa). Three different MWCNT-PEI conjugates are synthesized and evaluated for their condensation ability, viability, size and zeta potential properties. The transfection efficiency of the functionalized MWCNTs is evaluated using luciferase assay and flow cytometry in a Neuroblastoma cell line. MWCNT-PEI (10 kDa) conjugate shows the highest transfection efficacy compared to others. For this carrier transfection efficacy exceeds the amount of PEI 25 kDa at similar carrier to plasmid weight ratio (C/P) and is around 3 times higher compared to PEI 25 kDa at C/P=0.8 as positive control regarding its high transfection efficiency and low cytotoxicity.

Cellular delivery of shRNA using aptamer-conjugated PLL-alkyl-PEI nanoparticles.

Introduction of an efficient gene delivery vector is still the main challenge of gene therapy. Both polyethylenimine (PEI) and poly(l-lysine) (PLL) comprise disadvantages which limited their application. To explore whether their deficiencies could be compensated by preparing copolymers consisting of both PLL and PEI, we generated several combinations of PLL-alkyl-PEI copolymers conjugated to aptamer and evaluated their both gene delivery efficiency and down-regulation of Bcl-XL, an anti-apoptotic gene, in lung cancer cell line. PLL was conjugated to either 10% or 50% of PEI by grafting different percentages of PEI to alkylated-PLL as core. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. According to transfection results, aptamer conjugated PLL-alkyl-10%-PEI (PLPE8%) was selected for further gene silencing study by plasmid shRNA. Decrease in Bcl-XL gene expression was estimated by both RT-PCR and western-blot experiments. The obtained results revealed that the new copolymers had appropriate nano-scale size (117-128 nm) even after aptamer conjugation (168-183 nm). Moreover, they exhibited increased transfection efficiencies by up to 1.8-5 folds and acceptable cytotoxicity. The apoptosis was induced in transfected cells by shRNA-aptamer-copolymer due to the down-regulation of mRNA and protein levels. This study suggested a new vector for targeted non-viral gene delivery with high transfection efficiency in lung cancer or pulmonary systems.

Nicotine content of domestic cigarettes, imported cigarettes and pipe tobacco in iran.

BACKGROUND: There are many different kinds of cigarettes and tobacco available in the market. Since nicotine content of various brands of cigarettes are very variable, therefore evaluation and comparison of nicotine content of different brands of cigarettes is important. The goal of the present study was to determine and compare nicotine content of various domestic and imported cigarettes available in the area. METHODS: Fourteen popular imported brands and nine popular domestic brands of cigarettes and three available brands of tobaccos were investigated for the amounts of nicotine content. Nicotine was extracted from each cigarette and tobacco samples and was analyzed by high performance liquid chromatography (HPLC) method. FINDINGS: The amount of nicotine in each cigarette was from 6.17 to 12.65 mg (1.23 ± 0.15 percent of tobacco weight in each cigarette) in domestic cigarettes. It was between 7.17-28.86 mg (1.80 ± 0.25 percent of tobacco weight in each cigarette) for imported cigarette, and between 30.08- 50.89 mg (3.82 ± 1.11 percent) for the pipe nicotine. There was significant difference in nicotine amount between imported and domestic brands of cigarettes. There was also no significant difference in nicotine content between light and normal cigarettes in imported brands. CONCLUSION: Nicotine content of all tested cigarettes, imported and domestic brands, were higher than the international standard.