Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA.

To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100-280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18). More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

Nano-composite hydrogels of Cu-Apa micelles for anti-vasculogenic mimicry.

Vasculogenic mimicry (VM) describes the phenomenon whereby fluid-conducting vessels are formed by highly invasive tumour cells, which supply blood to tumours during their early growth stages. Single antiangiogenic agents have limited inhibitory effects on VM, therefore, a multi-pathway anti-VM strategy is required. In this study, Apatinib (Apa) was coordinated with Cu2+ to form a Cu-Apa copper complex. The latter was loaded into oligo-hyaluronic acid (HA) polymeric micelles (HA-Chol) and subsequently embedded in Astragalus polysaccharide-based in situ hydrogels (APsGels) to generate Cu-Apa/HA-Chol@APsGels. In this system, Cu-Apa exerts the combined effects of Cu2+ and Apa to inhibit VM; HA-Chol micelles achieve targeted drug delivery and enhance endocytosis efficiency; APsGels realise sustained release of the drugs to ensure an anti-VM effect. This system demonstrated improved VM inhibition with low cytotoxicity and high biocompatibility, wound healing, and transwell invasion in three-dimensional cell cultured VM. Moreover, this system significantly inhibited VM formation and melanoma growth in a mouse tumour transplantation model. This study provides an effective strategy for inhibiting VM.

A nano-targeted co-delivery system based on gene regulation and molecular blocking strategy for synergistic enhancement of platinum chemotherapy sensitivity in ovarian cancer.

Ovarian cancer (OC) has a low five-year survival rate, mainly because of its drug resistance to chemotherapy. It is the key to reverse drug resistance to combine multiple sensitization pathways to play a synergistic role. A nano scaled targeted co-delivery system (P123-PEI-G12, PPG) modified by bifunctional peptide tLyP-1-NLS (G12) was fabricated by using Pluronic P123 conjugated with low molecular weight polyethyleneimine (PEI). This delivery system can co-delivery Olaparib (Ola) and p53 plasmids to synergistically enhance the sensitivity of OC to platinum-based chemotherapy. P53@P123-PEI-G2/Ola (Co-PPGs) can achieve efficient tumor accumulation and cellular internalization through G12-mediated targeting. Co-PPGs then break down in the tumor cells, releasing the drug. Co-PPGs significantly enhanced the sensitivity of cisplatin (DDP) in platinum-resistant ovarian cancer (PROC) and synergistically inhibited the proliferation of PROC in vitro and in vivo. The sensitizing and synergistic effects of Co-PPGs were related to the activation of p53, inhibition of poly-ADP-ribose polymerase (PARP) and p-glycoprotein (P-gp) expression. This work provides a promising strategy for the effective treatment of PROC.

Mitochondrial targeted hierarchical drug delivery system based on HA-modified liposomes for cancer therapy.

Chemotherapy targeting mitochondrial is a faster and more sensitive anti-tumor therapy strategy. In this study, a hierarchical drug delivery system HA-GDT-Lip was constructed by coupling glycyrrhetinic acid (GA), triphenylphosphine (TPP), and doxorubicin (DOX), encapsulating them in cationic liposomes (CLs), then coating the surface of CLs with HA. HA-GDT-Lip nanoparticles can be accumulated in tumor tissue through the EPR effect, then achieve tumor cell-specific endocytosis mediated by the CD44 receptor, DOX can be successfully delivered into mitochondria through the combined action of GA and TPP. Physicochemical properties analysis showed that HA-GDT-Lip nanoparticles were uniform in size and spherical in shape. In vitro cell experiments showed that HA-GDT-Lip had high cell uptake efficiency and mitochondrial targeting ability. In addition, HA-GDT-Lip could induce MPTP opening and accelerate cell apoptosis. Meanwhile, HA-GDT-Lip showed excellent antitumor activity and in vivo safety in tumor-bearing nude mice. In conclusion, HA-GDT-Lip may serve as a promising mitochondrial delivery system to reduce the side effects of anticancer drugs and improve their antitumor efficacy.

Phragmites rhizoma polysaccharide-based nanocarriers for synergistic treatment of ulcerative colitis.

The purpose of this study was to construct Phragmites rhizoma polysaccharide-based nano-drug delivery systems (PRP2-SeNPs-H/Aza-Lips) for synergistically alleviating ulcerative colitis and to investigate the important roles of Phragmites rhizoma polysaccharide-based nanocarriers in PRP2-SeNPs-H/Aza-Lips. Phragmites rhizoma polysaccharide (PRP2) was isolated and used for the preparation of Phragmites rhizoma polysaccharide selenium nanoparticles with low selenium content (PRP2-SeNPs-L) and high selenium content (PRP2-SeNPs-H). Based on the electrostatic attraction between PRP2-SeNPs-H and azathioprine liposomes (Aza-Lips), PRP2-SeNPs-H/Aza-Lips were constructed for precise delivery of the model drug azathioprine (Aza) to colon lesions. Results showed that PRP2 significantly alleviated the clinical symptoms and colon tissue damage and down-regulated the levels of inflammatory factors in serum and colon, demonstrating beneficial effects on mice with ulcerative colitis. PRP2-SeNPs-L had better relieving effects on ulcerative colitis. Phragmites rhizoma polysaccharide-based nanocarriers may protect azathioprine liposomes against gastrointestinal digestion, enhance the therapeutic effects on ulcerative colitis, and significantly reduce liver damage from azathioprine, which helps to improve the efficacy and toxicity of clinical drugs.

Fe3+-Coordinated Multifunctional Elastic Nanoplatform for Effective in Vivo Gene Transfection.

The common phenomenon that the nonviral vectors have much lower transfection efficiency in vivo than in vitro greatly restricts their further developments and applications. Possible reasons are lacking targeting ability, elimination by the reticuloendothelial system (RES), and insufficient nuclear transport. Here, a novel, flexible, and deformable polymer Fe@PEI-R12 (tLyp-1-NLS) is reported for shortening the gap between in vitro and in vivo gene transfection efficiency. The amorphous network structure Fe@PEI with deformation ability acquired by coordination cross-linking of Fe3+ and low-molecular-weight polyethylenimine (LMW-PEI) constructs the core and serves as the gene reservoir, and it can squeeze out through RES filter holes when trapped in the spleen. The bifunctional peptide R12 provided tumor targeting and enhanced nuclear delivery ability. Additionally, the Fe3+ from Fe@PEI-R12 could trigger endogenous hydrogen peroxide (H2O2) decomposition to produce O2, thereby reducing the adverse effects of tumor hypoxia. It is demonstrated that the Fe@PEI-R12/pDNA complexes could pass through membrane filters, subsequently achieving long circulation time, and Fe@PEI-R12 had a tendency to accumulate in tumor tissue and mediate pGL3-control expression. Therefore, the multifunctional nanoplatform has the potential for effective in vivo gene delivery.

Modification of degradable nonviral delivery vehicle with a novel bifunctional peptide to enhance transfection in vivo.

AIM: To increase in vivo DNA transfection efficiency of a nonviral delivery vehicle, its tumor targeting and nuclear delivery ability was improved. MATERIALS & METHODS: A novel bifunctional peptide tLyP-1-NLS (named K12) was prepared by coupling a tumor-targeting peptide (tLyP-1) with a nuclear localization signal (NLS), and then was used to modify a degradable polyethyleneimine (PEI) derivative called "N-octyl-N-quaternary chitosan (OTMCS)-PEI". The carrier OTMCS-PEI-K12 was characterized in terms of the physicochemical properties, in vitro gene transfection and antitumor effect in vivo. RESULTS: OTMCS-PEI-K12 showed good suitability, stability and transfection capacity in vitro on the premise of noncytotoxicity. OTMCS-PEI-K12/pING4 complexes induced extensive apoptosis of tumor tissues and shrunk the tumor volume of mice noticeably in vivo. CONCLUSION: This study offers a way to enhance in vivo transfection of a nonviral carrier.

A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo.

Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100-300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo.

The reflection of guided waves from simple dents in pipes.

Guided elastic waves have been anticipated as a rapid screening technique for pipe inspection. Dents occurring in pipes are a severe problem which may lead to the possibility of pipe failure. A study of the reflection characteristics of guided waves from dents of varying geometrical profile in pipes is investigated through experiments. Dented region is represented by a series of circumferential cross-sections and its geometric parameters are described by axial length and the maximum and minimum outer diameters. Both single and double sided dents are mechanically simulated in hollow aluminum pipes and then experimentally tested by exciting the longitudinal L(0,2) mode. A quantitative parameter, so-called deformation rate relating to the maximum and minimum outer diameters of the dents is defined to evaluate the effect of the extent of the deformation on the reflection. For both types of dents, it is shown that the reflection coefficients of the L(0,2) mode are all approximately a linear function of their respective deformation rates. Mode conversion occurs at the dents and reflections of the F(1,3) mode are identified. The results show that the amplitude of the reflected F(1,3) mode is generally higher when the dent has stronger non-axisymmetric features.

Polyethylenimine derivate conjugated with RGD-TAT-NLS as a novel gene vector.

To solve the contradiction between the cell toxicity and transfection efficiency of polyethylenimine (PEI) derivate in non-viral gene therapy, a novel gene vector, P123-PEI-R18 was synthesized by using biodegradable PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS. The particle size of P123-PEI-R18/DNA was around 100-250 nm. The gene vector could condense DNA at the weight ratio of 2 and protect plasmid DNA from being dissolved in the blood circulation. Importantly, the complexes exhibited lower cell toxicity and higher transfection efficiency contrasted with PEI 25 kDa in vitro. P123-PEI-R18 holds high potential as a safe and efficient gene vector.

Intracellular disassembly and localization of a new P123-PEI-R13/DNA complex.

The appropriate location and release of target gene is necessary for gene therapy. In our previous paper, a gene vector named P123-PEI-R13 has been successfully synthesized, and the physical characteristics and cellular trafficking of nanoparticle P123-PEI-R13/DNA has been explored explicitly, but little was known about its disassembly within cells. In order to investigate its intracellular disassembly, P123-PEI-R13/DNA complex was exposed to the different competitors (RNA, DNA, proteins) or different conditions of pH and osmolarity, DNA release was determined by gel electrophoresis. Meanwhile, confocal laser technology was used to locate the complex in cells. The results revealed that DNA, RNA and osmolarity could affect the stability of the complex obviously, especially RNA which exist in nucleus. In addition, the speed of DNA release decreased as the weight ratio of polymer increased. Images got by a confocal fluorescence microscope confirmed that after cell uptake, P123-PEI-R13 could translocate DNA into nucleus.

Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting.

BACKGROUND: A degradable polyethylenimine (PEI) derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. METHODS: We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS) to synthesize a degradable PEI derivative (OTMCS-PEI), and then used a bifunctional peptide, RGDC-TAT (49-57) called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. RESULTS: The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150-250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/µg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 µg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. CONCLUSION: OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

Degradable copolymer based on amphiphilic N-octyl-N-quatenary chitosan and low-molecular weight polyethylenimine for gene delivery.

BACKGROUND: Chitosan shows particularly high biocompatibility and fairly low cytotoxicity. However, chitosan is insoluble at physiological pH. Moreover, it lacks charge, so shows poor transfection. In order to develop a new type of gene vector with high transfection efficiency and low cytotoxicity, amphiphilic chitosan was synthesized and linked with low-molecular weight polyethylenimine (PEI). METHODS: We first synthesized amphiphilic chitosan - N-octyl-N-quatenary chitosan (OTMCS), then prepared degradable PEI derivates by cross-linking low-molecular weight PEI with amphiphilic chitosan to produce a new polymeric gene vector (OTMCS-PEI). The new gene vector was characterized by various physicochemical methods. We also determined its cytotoxicity and gene transfecton efficiency in vitro and in vivo. RESULTS: The vector showed controlled degradation. It was very stable and showed excellent buffering capacity. The particle sizes of the OTMCS-PEI/DNA complexes were around 150-200 nm with proper zeta potentials from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 2.25 U DNase I/µg DNA. Furthermore, they were resistant to dissociation induced by 50% fetal bovine serum and 1100 µg/mL sodium heparin. OTMCS-PEI revealed lower cytotoxicity, even at higher doses. Compared with PEI 25 KDa, the OTMCS-PEI/DNA complexes also showed higher transfection efficiency in vitro and in vivo. CONCLUSION: OTMCS-PEI was a potential candidate as a safe and efficient gene vector for gene therapy.

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector.

BACKGROUND: To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. METHODS: First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49-57), to yield a bifunctional peptide RGDC-Tat (49-57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvß3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. RESULTS: The vector showed controlled degradation, strong targeting specificity to αvß3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100-250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/µg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 µg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. CONCLUSION: P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy.