Systemic administration of human mesenchymal stromal cells infected with polymer-coated oncolytic adenovirus induces efficient pancreatic tumor homing and infiltration.

Oncolytic adenovirus (oAd)-mediated gene therapy is a promising approach for cancer treatment because of its cancer cell-restricted replication and therapeutic gene expression. However, systemic administration of oAd is severely restricted by their immunogenic nature and poor tumor homing ability, thus oAd cannot be utilized to treat disseminated metastases. In this study, human bone marrow-derived mesenchymal stromal cell (hMSCs) was used as a viral replication-permissive carrier for oAd with an aim to improve the systemic delivery of the virus to tumor tissues. To overcome the poor delivery of oAd into hMSCs, a relaxin (RLX)-expressing oncolytic Ad (oAd/RLX), which degrades dense tumor extracellular matrix of highly desmoplastic pancreatic cancer, was complexed with biodegradable polymer (poly (ethyleneimine)-conjugated poly(CBA-DAH); PCDP), generating oAd/RLX-PCDP complex. oAd/RLX-PCDP complex enhanced the internalization of oAd into hMSC, leading to superior viral production and release from hMSCs, along with high RLX expression. Furthermore, systemic administration of oAd/RLX-PCDP-treated hMSCs elicited more potent antitumor effect compared to naked oAd/RLX or oAd/RLX-treated hMSC in pancreatic tumor model. This potent antitumor effect of systemically administered oAd/RLX-PCDP-treated hMSCs was achieved by superior viral replication in tumor tissues than any other treatment group. In conclusion, these results demonstrate that hMSCs are effective carriers for the systemic delivery of oAd to tumor sites and treatment of pancreatic cancer.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied.

Stent linker effect in a porcine coronary restenosis model.

In this study, we aimed to evaluate the mechanical effects of different stent linker designs on in-stent restenosis in porcine coronary arteries. We fabricated stents with an open-cell structure composed of nine main cells and three linker structures in model 1 (I-type), model 2 (S-types) and model 3 (U-types)) as well as Model 4, which is similar to a commercial bare metal stent design. The stent cells were 70 mm thick and wide, with a common symmetrical wave pattern. As the radial force increased, the number of main cells increased and the length of linker decreased. Radial force was higher in model 1, with a linear I-linker, than in models with S- or U-linkers. The flexibility measured by three-point bending showed a force of 1.09 N in model 1, 0.35 N in model 2, 0.19 N in model 3, and 0.31 N in model 4. The recoil results were similar in all models except model 4 and were related to the shape of the main cells. The foreshortening results were related to linker shape, with the lowest foreshortening observed in model 3 (U-linker). Restenosis areas in the porcine restenosis model 4 weeks after implantation were 35.4 ± 8.39% (model 1), 30.4 ± 7.56% (model 2), 40.6 ± 9.87% (model 3) and 45.1 ± 12.33% (model 4). In-stent restenosis rates measured by intravascular ultrasound (IVUS) and micro-computed tomography (micro-CT) showed similar trends as percent area stenosis measured by micro-CT. Model 2, with optimized flexibility and radial force due to its S-linker, showed significantly reduced restenosis in the animal model compared to stents with different linker designs. These results suggest that the optimal stent structure has a minimum radial force for vascular support and maximum flexibility for vascular conformability. The importance of the effects of these differences in stent design and their potential relationship with restenosis remains to be determined.

Target gene delivery from targeting ligand conjugated chitosan-PEI copolymer for cancer therapy.

In this study, we designed a novel carrier which was having low cytotoxicity, site-specific target function, and high transfection efficiency using low molecular weight water soluble O-carboxymethyl chitosan (OCMCh), branched low molecular weight poly(ethyleneimine) (bPEI), and targeting ligand (epitope type, HER-2/neu). OCMCh/bPEI/targeting ligand, HPOCP copolymer, and targeting ligand-modified polyamphoteric polymer, and were prepared by chemical reaction and characterized by (1)H NMR and FT-IR. The binding affinity, protecting efficiency, and releasing ability of gene/HPOCP polyplex were confirmed by gel retardation assay. The pDNA(pEGFP)/HPOCP polyplexes showed high gene transfection efficiency in HCT 119 cell. In addition, siRNA/HPOCP polyplexes formed spherical shape and have particle sizes from 100 to 300nm. The siRNA/HPOCP polyplexes have lower cytotoxicity than PEI in the all of siRNA concentrations ranging from 0 to 2µg/µL in HEK 293 cells. The cell viability of siRNA/HPOCP polyplexes was performed in SK-Br3 cells with VEGF siRNA or BCL2 siRNA. In addition, confocal laser-scanning microscopy and flow cytometry assay were performed for cellular localization and cellular uptake efficiency of siRNA/HPOCP polyplexes. The results of the present study demonstrate that HPOCP copolymer is a good candidate as gene delivery carriers for gene delivery system or gene therapy.

Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility.

VEGF therapeutic gene delivery using dendrimer type bio-reducible polymer into human mesenchymal stem cells (hMSCs).

The therapeutic potential of mesenchymal stem cells (MSCs) has garnered great attention in the expansive diversity of biomedical research. Despite this broad interest in stem cells, limited incorporation and poor viability are major disadvantages for accomplishing therapeutic success in the field of hMSC-based cell therapy, and an optimal approach for hMSC-based cell therapy using non-viral vectors has not been established. Hence, we examined the possibility of performing gene therapy using the biodegradable polymeric non-viral vector Arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP)-conjugated poly (amidoamine) (PAMAM) dendrimer (PAM-ABP) in hMSCs. PAM-ABP formed compact nanosized polyplexes and showed low cytotoxicity compared to bPEI 25k and Lipofectamine® 2000 in hMSCs. Although the cellular uptake was similar, the transfection efficiency and VEGF expression of PAM-ABP using gWiz-Luc and pß-VEGF were higher than those of the control groups. Although hMSCs were transfected, their stem cell characteristics were retained. Our results suggest that PAM-ABP has the ability to deliver a therapeutic gene in hMSCs.

Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: In vitro and in vivo evaluation.

In this study, we report the development of a novel, redox-sensitive chitosan-based targeted drug delivery system, containing two drugs. We determined whether the synthesized polymeric micelles (HPTOC-DOX) were suitable as a drug carrier. The formation of HPTOC-DOX micelles was confirmed by (1)H NMR. HPTOC-DOX formed micelles of approximately 151.9-311.2nm in size in aqueous solution. Analysis of the drug release profile of HPTOC-DOX in different pH conditions (pH 5.2, 6.2, and 7.4) indicated that DOX was released from HPTOC-DOX micelles at acidic pH (5.2 or 6.2), while almost no DOX was released at pH 7.4. In vitro cell cytotoxicity and hemolysis assays indicated that HPTOC-DOX micelles safely deliver anti-cancer drugs and decrease the cytotoxicity of DOX. In vitro anti-cancer activity assays, confocal laser scanning microscopy analysis of SK-BR-3 cells, and in vivo anti-tumor activity in SK-BR-3-derived tumor-bearing mice were used to evaluate synergistic drug effects and the effect of the targeting peptide (anti-human epidermal growth factor receptor 2 [HER2] target peptide, epitope form; LTVSPWY) on receptor-mediated endocytosis.

Evaluation of Histidylated Arginine-Grafted Bioreducible Polymer To Enhance Transfection Efficiency for Use as a Gene Carrier.

To increase cellular uptake and endosomal escape efficiency, various methods have been studied to efficiently deliver plasmid DNA (pDNA) into the cell. Here, we designed a histidylated arginine-grafted bioreducible polymer (HABP) as a nonviral gene carrier using different ratios of histidine and arginine-grafted bioreducible poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)), known as ABP, to increase cellular uptake and endosomal escape efficiency. HABPs consist of arginine (cell penetrating functionality), histidine (endosome buffering functionality), and a disulfide bond backbone (bioreducible functionality in cytoplasm). These components result in the following: (1) polyplexes are easily taken up by cells, (2) polyplexes can easily escape from the endosome into the cytosol, and (3) pDNA can dissociate from polyplexes in reducing environments such as the cytoplasm. HABPs showed increased buffering capacity over histidine-ungrafted ABP, and HABPs formed nanosized polyplexes with pDNA. These polyplexes were about 90 nm in size and had positive charges of about of 30-40 mV. HABPs/pDNA polyplexes showed enhanced transfection efficiency and no significant cytotoxicity in comparison with polyethylenimine 25 kDa (PEI 25k), histidine-ungrafted ABP, and Lipofectamine (commercial reagent) in human cervical carcinoma (HeLa), rat cardiomyocytes (H9C2), and colon carcinoma (CT26) cells.

Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy.

Recently, adenovirus (Ad) has been utilized as a viral vector for efficient gene delivery. However, substantial immunogenicity and toxicity have obstructed oncolytic Ad's transition into clinical studies. The goal of this study is to generate an adenoviral vector complexed with multidegradable bioreducible core-cross-linked polyethylenimine (rPEI) polymer that has low immunogenicity and toxicity while having higher transduction efficacy and stability. We have synthesized different molecular weight rPEIs and complexed with Ad at varying molar ratios to optimize delivery of the Ad/polymer complex. The size and surface charge of Ad/rPEIs were characterized. Of note, Ad/rPEIs showed significantly enhanced transduction efficiency compared to either naked Ad or Ad/25 kDa PEI in both coxsackievirus and adenovirus receptor (CAR) positive and negative cancer cells. The cellular uptake result demonstrated that the relatively small size of Ad/16 kDa rPEIs (below 200 nm) was more critical to the complex's internalization than its surface charge. Cancer cell killing effect and viral production were significantly increased when oncolytic Ad (RdB/shMet, or oAd) was complexed with 16 kDa rPEI in comparison to naked oAd-, oAd/25 kDa PEI-, or oAd/32 kDa rPEI-treated cells. This increased anticancer cytotoxicity was more readily apparent in CAR-negative MCF7 cells, implying that it can be used to treat a broad range of cancer cells. Furthermore, A549 and HT1080 cancer cells treated with oAd/16 kDa rPEI had significantly decreased Met and VEGF expression compared to either naked oAd or oAd/25 kDa PEI. Overall, these results demonstrate that shMet expressing oncolytic Ad complexed with multidegradable bioreducible core-cross-linked PEI could be used as efficient and safe cancer gene therapy.

Evaluation of dendrimer type bio-reducible polymer as a siRNA delivery carrier for cancer therapy.

Small interfering ribonucleic acid (siRNA), 20-25 base pairs in length, can interfere with the expression of specific genes. Recently, many groups reported the therapeutic intervention of siRNA in various cancer cells. In this study, dendrimer type bio-reducible polymer (PAM-ABP) which was synthesized using arginine grafted bio-reducible poly(cystaminebisacrylamide-diaminohexane) (ABP) and polyamidoamine (PAMAM) was used to deliver anti-VEGF siRNA into cancer cell lines including human hepatocarcinoma (Huh-7), human lung adenocarcinoma (A549), and human fibrosarcoma (HT1080) cells and access their potential as a siRNA delivery carrier for cancer therapy. PAM-ABP and siRNA formed polyplexes with average diameter of 116 nm and charge of around +24.6 mV. The siRNA in the PAM-ABP/siRNA polyplex was released by 5mM DTT and heparin. VEGF gene silencing efficiency of PAM-ABP/siRNA polyplexes was shown to be more effective than PEI/siRNA polyplexes in three cell lines with the following order HT1080>A549>Huh-7.

Antimicrobial action of water-soluble ß-chitosan against clinical multi-drug resistant bacteria.

Recently, the number of patients infected by drug-resistant pathogenic microbes has increased remarkably worldwide, and a number of studies have reported new antibiotics from natural sources. Among them, chitosan, with a high molecular weight and α-conformation, exhibits potent antimicrobial activity, but useful applications as an antibiotic are limited by its cytotoxicity and insolubility at physiological pH. In the present study, the antibacterial activity of low molecular weight water-soluble (LMWS) α-chitosan (α1k, α5k, and α10k with molecular masses of 1, 5, and 10 kDa, respectively) and ß-chitosan (ß1k, ß5k, and ß10k) was compared using a range of pathogenic bacteria containing drug-resistant bacteria isolated from patients at different pH. Interestingly, ß5k and ß10k exhibited potent antibacterial activity, even at pH 7.4, whereas only α10k was effective at pH 7.4. The active target of ß-chitosan is the bacterial membrane, where the leakage of calcein is induced in artificial PE/PG vesicles, bacterial mimetic membrane. Moreover, scanning electron microscopy showed that they caused significant morphological changes on the bacterial surfaces. An in vivo study utilizing a bacteria-infected mouse model found that LMWS ß-chitosan could be used as a candidate in anti-infective or wound healing therapeutic applications.

Anticancer effect of gene/peptide co-delivery system using transferrin-grafted LMWSC.

A series of ternary complex was designed to deliver psiRNA-bcl2 and (KLA)4 peptide into cancer cells for cancer therapy. The delivered psiRNA-bcl2 induced gene-silencing in a nucleus of cancer cells, while (KLA)4 peptide inhibited cancer growth via mitochondrial apoptosis, indicating that the ternary complexes exerted very strong synergistic effects on cancer growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide simultaneously. The ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119. The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32-64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165-248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3-4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The useful complexes for gene and peptide delivery had high anticancer activities via a synergistic effect due to co-operative action of psiRNA and (KLA)4 peptide in HCT119 cells.

Evaluation of polyethylene glycol-conjugated novel polymeric anti-tumor drug for cancer therapy.

A novel polymeric prodrug (PXPEG) was prepared to enhance the solubility of an anti-cancer drug, paclitaxel, in aqueous solutions and decrease the cytotoxicity by PEGylation, which means PEG attached to another molecule. In addition, the targeting ligand, transferrin (TF), was modified to PXPEG to enhance the therapeutic efficacy. The targeting ligand-modified PXPEG (TFPXPEG) was examined by (1)H-NMR to confirm the successful synthesis. The synthesized TFPXPEG had better solubility than the free drug against aqueous solution. The particle size of TFPXPEG was approximately 197.2nm and it had a spherical shape. The MTT assay showed that the anti-tumor efficiency of TFPXPEG was better than that of TF-unmodified PXPEG. In the KB tumor-bearing mouse model, the tumor volume of TFPXPEG treated groups was decreased dramatically by more than 2 fold or 3 fold compared to the PBS or PXPEG treated groups. The in vitro and in vivo evaluation showed that TFPXPEG had better efficacy than that of PXPEG due to the targeting effect of targeting ligands, such as TF.

Immunoadjuvant efficacy of N-carboxymethyl chitosan for vaccination via dendritic cell activation.

To induce humoral- and cell-mediated immune responses in protein-based vaccinations, immunoadjuvants are needed. In this study, we investigated the immunoadjuvant effect of N-carboxymethyl chitosan (N-CMCh) on the vaccination system through dendritic cell activation. The results showed that N-CMCh nanoparticle enhanced the secretion of cytokines (interleukin [IL]-6, IL-12p70, and tumor necrosis factor-α) and antigen uptake in bone marrow-derived dendritic cells. This activated antigen-specific Th1 cell responses, including IL-2 and interferon-gamma production and proliferation. In addition, N-CMCh delayed the dissemination of ovalbumin (OVA) from an injection site in female C57BL/6 mice. As a result, in the subcutaneous vaccination, OVA-N-CMCh nanoparticles enhanced both humoral and cell-mediated vaccine responses.

Branched polyethylenimine-grafted-carboxymethyl chitosan copolymer enhances the delivery of pDNA or siRNA in vitro and in vivo.

To generate a good carrier for gene transfection, O-carboxymethyl chitosan-graft-branched polyethylenimine (OCMPEI) copolymers were synthesized by increasing the weight percentage of branched polyethylenimine conjugated to the carboxyl groups of O-carboxymethyl chitosan. These spherical polyplexes with plasmid deoxyribonucleic acid (pDNA) or small interfering ribonucleic acid (siRNA) had diameters of ~200-300 nm or ~10-25 nm, respectively, and displayed significant transfection efficiency in normal and tumor cells. In particular, expression of green fluorescent protein (GFP) following pDNA transfection was effectively suppressed by delivery of GFP-specific siRNA with the same copolymer. The optimized copolymer and polyplexes were nontoxic in vitro and in vivo. The use of endocytosis inhibitors to investigate the mechanisms of transfection of the polyplexes suggested the involvement of macropinocytosis. An in vivo study in mice showed excellent GFP expression in the lung, kidney, and liver. The results demonstrated that the OCMPEI copolymer prepared in this study is a promising carrier for in vitro and in vivo gene delivery applications.

Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery.

Transferrin/PEG/O-carboxymethyl chitosan/fatty acid/paclitaxel (TPOCFP) micelles were tested for suitability as a drug carrier characterized by low cytotoxicity, sustained release, high cellular uptake, and site-specific targeted delivery of hydrophobic drugs. Characterization, drug content, encapsulation efficiency, and in vitro drug release were investigated. When the feeding amount of paclitaxel (PTX) was increased, the drug content increased, but loading efficiency decreased. TPOCFP micelles had a spherical shape, with a particle size of approximately 140-649 nm. In vitro cell cytotoxicity and hemolysis assays were conducted to confirm the safety of the micelles. Anticancer activity and confocal laser scanning microscopy (CLSM) were used to confirm the targeting efficiency of target ligand-modified TPOCFP micelles. Anticancer activity and CLSM results clearly demonstrated that transferrin-modified TPOCFP micelles were quickly taken up by the cell. The endocytic pathway of TPOCFP micelles was analyzed by flow cytometry, revealing transfection via receptor-mediated endocytosis. These results suggest that PTX-encapsulated TPOCFP micelles may be used as an effective cancer-targeting drug delivery system for chemotherapy.