Aptamer-functionalized mesenchymal stem cells-derived exosomes for targeted delivery of SN38 to colon cancer cells.

Objectives: Known as natural nanovesicles, exosomes have attracted increased attention as biocompatible carriers throughout recent years, which can provide appropriate sources for incorporating and transferring drugs to desired cells in order to improve their effectiveness and safety. Materials and Methods: This study implicates the isolation of mesenchymal stem cells from adipocyte tissue (ADSCs) to acquire a proper amount of exosomes for drug delivery. As the exosomes were separated by ultracentrifugation, SN38 was entrapped into ADSCs-derived exosomes through the combination method of incubation, freeze-thaw, and surfactant treatment (SN38/Exo). Then, SN38/Exo was conjugated with anti-MUC1 aptamer (SN38/Exo-Apt), and its targeting ability and cytotoxicity towards cancer cells were investigated. Results: Encapsulation efficiency of SN38 into exosomes (58%) was significantly increased using our novel combination method. Furthermore, the in vitro results were indicative of the great cellular uptake of SN38/Exo-Apt and its significant cytotoxicity on Mucin 1 overexpressing cells (C26 cancer cells) without noticeable cytotoxicity on normal cells (CHO cells). Conclusion: The results propose that our approach developed an efficient method for loading SN38 as a hydrophobic drug into exosomes and decorating them with MUC1 aptamer against Mucin 1 overexpressing cells. So, SN38/Exo-Apt could be considered a great platform in the future for the therapy of colorectal cancer.

Targeted delivery of galbanic acid to colon cancer cells by PLGA nanoparticles incorporated into human mesenchymal stem cells.

Objective: The aim of this study was to investigate the efficacy of mesenchyme stem cells (MSCs) derived from human adipose tissue (hMSCs) as carriers for delivery of galbanic acid (GBA), a potential anticancer agent, loaded into poly (lactic-co-glycolic acid) (PLGA) nanoparticles (nano-engineered hMSCs) against tumor cells. Materials and Methods: GBA-loaded PLGA nanoparticles (PLGA/GBA) were prepared by single emulsion method and their physicochemical properties were evaluated. Then, PLGA/GBA nanoparticles were incorporated into hMSCs (hMSC/PLGA-GBA) and their migration ability and cytotoxicity against colon cancer cells were investigated. Results: The loading efficiency of PLGA/GBA nanoparticles with average size of 214±30.5 nm into hMSCs, was about 85 and 92% at GBA concentration of 20 and 40 µM, respectively. Nano-engineered hMSCs showed significant higher migration to cancer cells (C26) compared to normal cells (NIH/3T3). Furthermore, nano-engineered hMSCs could effectively induce cell death in C26 cells in comparison with non-engineered hMSCs. Conclusion: hMSCs could be implemented for efficient loading of PLGA/GBA nanoparticles to produce a targeted cellular carrier against cancer cells. Thus, according to minimal toxicity on normal cells, it deserves to be considered as a valuable platform for drug delivery in cancer therapy.

Thymoquinone-loaded mesenchymal stem cell-derived exosome as an efficient nano-system against breast cancer cells.

Objectives: Exosomes became the subject of extensive research in drug delivery approach due to their potential applicability as therapeutic tools for cancer therapy. Thymoquinone (Tq) is an anti-cancer agent due to its great anti-proliferative effect. However, poor solubility and weak bioavailability restrict its therapeutic applications. In this study, exosomes secreted from human adipocyte-derived mesenchymal stem cells (AdMSCs) were isolated and the efficacy of a novel encapsulation method for loading of Tq was investigated. Finally, the cytotoxic effect of Tq incorporated exosomes against cancer cells was evaluated. Materials and Methods: Exosomes secreted from AdMSCs were isolated via ultracentrifugation and characterized by electron microscopy and western blotting. Then, through a novel encapsulation approach, Tq was loaded into exosomes by the combination of three methods including incubation, freeze-thawing, and surfactant treatment. Then, the encapsulation efficiency, in vitro cellular uptake, and cytotoxicity of Tq incorporated exosomes (Tq@EXOs) in MCF7 and L929 cells were estimated. Results: Tq loading into exosomes through our novel method caused a significant improvement in encapsulation efficiency of about 60%. The fluorescent microscopy and flow cytometry outcomes indicated the efficient uptake of Tq@EXOs-FITC by cells throughout 4 hr. Furthermore, MTT results displayed the ability of Tq@EXOs in effectively decreasing the cell viability of MCF7 without causing any obvious cytotoxicity on L929 as normal cells. Conclusion: The results suggest that our approach provides effective loading of Tq into exosomes which offer a valuable and safe platform for drug delivery to cancer cells thus having a great potential for clinical studies.

Development of targeted gene delivery system based on liposome and PAMAM dendrimer functionalized with hyaluronic acid and TAT peptide: In vitro and in vivo studies.

The development of gene delivery systems is essential to improve their transfection efficiency and cytotoxicity. Combination of lipid and polymeric nanoparticles with the characteristics of both systems have been considered as a next-generation gene delivery platform. In the current study, we designed a novel and efficient targeted gene delivery system based on liposome and PAMAM dendrimer in cancer cells. Two polymeric formulations containing polyamidoamine-TAT (PAMAM-TAT) and PAMAM-TAT-Hyaluronic acid (HA) and two lipopolymeric carriers including PAMAM-TAT-Liposome and PAMAM-TAT-HA-Liposome were complexed with the enhanced green fluorescent protein (EGFP) plasmid and then evaluated in terms of physicochemical characteristics. The cytotoxicity and transfection efficiency of these synthetized carriers were accomplished against murine colon carcinoma cell line (C26). The biodistribution of polyplexes and lipoployplexes was also evaluated in the C26 tumor bearing mice. The results showed no significant toxicity for all designed nanoparticles (NPs) in C/P4. The highest gene expression was observed using lipopolyplex PAMAM-TAT-HA-Liposome in C/P4 (ratio polymer/DNA; wt/wt). Biodistribution study demonstrated more aggregation of targeted lipopolyplex in tumor cells than other nanoparticles (NPs). It could be concluded that the developed targeted lipopolymeric complex could serve as promising nanotherapeutic system for gene therapy.

Bromelain Loaded Lipid-Polymer Hybrid Nanoparticles for Oral Delivery: Formulation and Characterization.

Bromelain (Br), a mixture of proteolytic enzymes from pineapple (Ananas comosus), has various therapeutic potentials; however, its low bioavailability has limited the clinical applications specifically in oral delivery as the most common convenient used route of administration. In the present study, a lipopolymeric nanoparticle (NP) containing Br was developed to enhance its stability and oral delivery efficiency. Firstly, Br was loaded into poly (D, L-lactide-co-glycolide acid) (PLGA) and PLGA-phosphatidylcholine (PLGA-PC) NPs using double emulsion solvent evaporation technique. Then, Br integrity and activity were investigated using SDS-PAGE and gelatin test. The stability and release profile of Br from synthetized NPs were evaluated at different pH values of the digestive system. Furthermore, cytotoxicity, cellular uptake, and the amount of Br passage from Caco-2 cells were explored. The results showed PLGA-PC-Br NPs had higher encapsulation efficiency (83%) compared to PLGA-Br NPs (50%). In addition, this NP showed more Br released in neutral (20.36%) and acidic (34%) environments compared to PLGA-Br NPs after 5 days. The delay in the release of Br from PLGA-PC-Br NPs versus the faster release of Br from PLGA-Br formulation could assure that an appropriate concentration of Br has reached the intestine. Intestinal absorption study demonstrated that lipid polymer NPs were able to pass through Caco-2 cells about 1.5 times more (98.4%) than polymeric NPs (70%). In conclusion, PLGA-PC NPs would be considered as a promising lipid-polymer nanocarrier for effective intestinal absorption of Br.

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.

Optimization of PLGA formulation containing protein or peptide-based antigen: Recent advances.

Protein or peptide-based antigens are the most promising forms to generate custom protective immune responses for clinical applications. Over the last decades, poly(lactic-co-glycolic acid) (PLGA) as a biodegradable polymer has gained more attention for delivery of protein and peptide. Besides many appropriate characteristics, to improve its properties to overcome some obstacles such as release profile and it is important instability of antigen during both encapsulation and storage. Therefore, optimized procedures conditions require to be used to maintain the integrity of protein structure under several stress factors in formulation process. In this review article, the properties of PLGA particles, their preparation techniques and strategies for improvement of protein stability during encapsulation into PLGA, release from particle and storage as well as stabilization approaches were summarized. © 2018 Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part A: 106A: 2540-2551, 2018.

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.

Co-delivery of Dual Toll-Like Receptor Agonists and Antigen in Poly(Lactic-Co-Glycolic) Acid/Polyethylenimine Cationic Hybrid Nanoparticles Promote Efficient In Vivo Immune Responses.

Strategies to design delivery vehicles are critical in modern vaccine-adjuvant development. Nanoparticles (NPs) encapsulating antigen(s) and adjuvant(s) are promising vehicles to deliver antigen(s) and adjuvant(s) to antigen-presenting cells (APCs), allowing optimal immune responses against a specific pathogen. In this study, we developed a novel adjuvant delivery approach for induction of efficient in vivo immune responses. Polyethylenimine (PEI) was physically conjugated to poly(lactic-co-glycolic) acid (PLGA) to form PLGA/PEI NPs. This complex was encapsulated with resiquimod (R848) as toll-like receptor (TLR) 7/8 agonist, or monophosphoryl lipid A (MPLA) as TLR4 agonist and co-assembled with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN) as TLR9 agonist to form a tripartite formulation [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The physicochemical characteristics, cytotoxicity and cellular uptake of these synthesized delivery vehicles were investigated. Cellular viability test revealed no pronounced cytotoxicity as well as increased cellular uptake compared to control groups in murine macrophage cells (J774 cell line). In the next step, PLGA (MPLA or R848)/PEI (CpG ODN) were co-delivered with ovalbumin (OVA) encapsulated into PLGA NPs to enhance the induction of immune responses. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokine (IFN-γ, IL-4, and IL-1ß) secretion and antibody (IgG1, IgG2a) production. Robust and efficient immune responses were achieved after in vivo administration of PLGA (MPLA or R848)/PEI (CpG ODN) co-delivered with OVA encapsulated in PLGA NPs in BALB/c mice. Our results demonstrate a rational design of using dual TLR agonists in a context-dependent manner for efficient nanoparticulate adjuvant-vaccine development.

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.

Current Strategies in the Modification of PLGA-based Gene Delivery System.

Successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and genes efficiently. This formulation has several advantages in comparison with other formulations including improvement in solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as a gene carrier, there exist many challenges. PLGA NPs could protect the encapsulated DNA from in vivo degradation but the DNA release is slow and the negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce cytotoxicity, to enhance delivery efficiency and to target specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for the modification of PLGA particles applied in gene therapy.

Induction of a balanced Th1/Th2 immune responses by co-delivery of PLGA/ovalbumin nanospheres and CpG ODNs/PEI-SWCNT nanoparticles as TLR9 agonist in BALB/c mice.

To develop effective and safe vaccines with reduced dose of antigen and adjuvant, intelligent delivery systems are required. Many delivery systems have been developed to enhance the biological activity of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) as both immunotherapeutic agents and vaccine adjuvants. In this study we designed a novel CpG ODN delivery system based on single-walled carbon nanotube (SWCNT) functionalized with polyethylenimine (PEI) and alkylcarboxylated PEI (AL-PEI). The physicochemical characteristics, cytotoxicity and cellular uptake studies of these carriers were performed. All carriers were conjugated with CpG ODN followed by co-delivery with ovalbumin (OVA) encapsulated into poly (lactic-co-glycolic acid) nanospheres (PLGA NSs) to enhance the induction of immune responses. The effect of these formulations on antibody (IgG1, IgG2a) and cytokine (IL-1ß, IFN-γ, IL-4) production was evaluated in an in vivo experiment. The results showed that all nano-adjuvant formulations had a strong influence in up-regulation of IFN-γ and IL-4 in parallel with high IgG1-IgG2a isotype antibody titers in mice. In particular, SWCNT-AL-PEI nano-adjuvant formulation generated a balanced Th1 and Th2 immune response with more biased toward Th1 response without exhibiting any inflammatory and toxic effects. Therefore this nano-adjuvant formulation could be used as an efficient prophylactic immune responses agent.

Multivariate statistical techniques for the assessment of seasonal variations in surface water quality of pasture ecosystems.

This study investigates the applicability of multivariate statistical techniques including cluster analysis (CA), discriminant analysis (DA), and factor analysis (FA) for the assessment of seasonal variations in the surface water quality of tropical pastures. The study was carried out in the TPU catchment, Kuala Lumpur, Malaysia. The dataset consisted of 1-year monitoring of 14 parameters at six sampling sites. The CA yielded two groups of similarity between the sampling sites, i.e., less polluted (LP) and moderately polluted (MP) at temporal scale. Fecal coliform (FC), NO3, DO, and pH were significantly related to the stream grouping in the dry season, whereas NH3, BOD, Escherichia coli, and FC were significantly related to the stream grouping in the rainy season. The best predictors for distinguishing clusters in temporal scale were FC, NH3, and E. coli, respectively. FC, E. coli, and BOD with strong positive loadings were introduced as the first varifactors in the dry season which indicates the biological source of variability. EC with a strong positive loading and DO with a strong negative loading were introduced as the first varifactors in the rainy season, which represents the physiochemical source of variability. Multivariate statistical techniques were effective analytical techniques for classification and processing of large datasets of water quality and the identification of major sources of water pollution in tropical pastures.