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.

Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111In Auger Electrons as DNA-Targeting Radioimmunoconjugate.

BACKGROUND: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development. OBJECTIVES: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. METHODS: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA- NLS-trastuzumab were labeled with 111In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for 111In, 111In-DTPA-trastuzumab and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection. RESULTS: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1, was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA- NLS-trastuzumab. CONCLUSION: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate.

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.

Design of a Novel Nucleus-Targeted NLS-KALA-SA Nanocarrier to Delivery Poorly Water-Soluble Anti-Tumor Drug for Lung Cancer Treatment.

In this study, we designed a novel nucleus-targeted nanocarrier (NLS-KALA-SA, NKSN) consisting of Kala peptide (KALA), nuclear localization signal (NLS) and stearic acid (SA) using Fmoc solid phase synthesis method. We chose Curcumin (CUR), Paclitaxel (PTX), Ginsenoside compound K(CK) as models of poorly water-soluble antitumor drugs, The drugs loaded NLS-KALA-SA nanoparticles (CUR/NKSN, PTX/NKSN, CK/NKSN) were obained by the dialysis method, their physicochemical properties were determined and antitumor activity were evaluated. The NLS-KALA-SA nanoparticles were spherical shaped with an average size of 76.4 ± 7.6 mm and a zeta potential of 43.7 ± 5.8 mV. The drug-loaded NLS-KALA-SA nanoparticles were above 86.1% and 17.1% in entrapment efficiency and drug loading capacity, and had sustained drug release behavior. Biodistribution and cellular uptake study exhibited that PTX/NKSN mainly distributed in tumor site of A549-bearing mice, and coumarin-6(C6) loaded NLS-KALA-SA nanoparticle (C6/NKSN) was predominantly accumulated in the nucleus of A549 cells. Western blot analysis indicated that PTX/NKSN could more remarkably inhibit Bcl-2 expression and enhance the expression of Bax and Caspase-3 as compared to the controls in A549 cells. Cell apoptosis and antitumor activity study showed that PXT/NKSN could more obviously induce apoptosis of A549 cells compared with free PXT, the PTX/NKSN administration was more effective than free PTX for lung cancer treatment and displayed mild toxicity in A549-bearing mice. The results demonstrates that the NLS-KALA-SA nanoparticles system could enhance the antitumor effects of the encapsulated drug and reduce tissue toxicity due to its long circulating properties and tumor targeting, which might provide a promising strategy for lung cancer treatment.

Drug-resistant AML cells and primary AML specimens are killed by 111In-anti-CD33 monoclonal antibodies modified with nuclear localizing peptide sequences.

UNLABELLED: Multidrug resistance (MDR) is a major challenge to the successful treatment of acute myeloid leukemia (AML). Our purpose was to determine whether (111)In-HuM195 anti-CD33 antibodies modified with peptides harboring nuclear localizing sequences (NLS) could kill drug-resistant AML cell lines and primary AML patient specimens expressing MDR transporters through the emission of Auger electrons. METHODS: HuM195, M195, and irrelevant mouse IgG (mIgG) were conjugated to 10+/-3 NLS peptides and then labeled with (111)In by diethylenetriaminepentaacetic acid substitution to a specific activity of 1-8 MBq/microg. The binding affinity of HuM195 and M195 was determined for HL-60 and mitoxantrone-resistant HL-60-MX-1 cells. Nuclear localization of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 was measured by subcellular fractionation. The antiproliferative effects of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 (2.5-250 kBq/well) on HL-60 and HL-60-MX-1 were studied using the WST-1 assay. Clonogenic survival of HL-60 and HL-60-MX-1 leukemic cells and 10 primary AML specimens with MDR phenotype (assessed by flow cytometry) was determined after exposure for 3 h at 37 degrees C to 2.5-250 mBq/cell of (111)In-NLS-HuM195, (111)In-HuM195, or (111)In-NLS-mIgG. Clonogenic survival versus the amount of radioactivity incubated with the cells (mBq/cell) was plotted, and the mean lethal amount of radioactivity and the lower asymptote of the curve (plateau) were determined. RESULTS: The (111)In-labeled anti-CD33 monoclonal antibodies HuM195 and M195 modified with NLS were efficiently routed to the nucleus of HL-60 cells and their mitoxantrone-resistant clone after CD33-mediated internalization. The following are the principal findings of our study: (111)In-NLS-HuM195 was more effective at killing HL-60 and HL-60-MX-1 cells than was (111)In-HuM195, a strong correlation between the specific activity of the (111)In-labeled radioimmunoconjugates and their cytotoxicity toward AML cells existed, and leukemic cells from patients were killed by (111)In-NLS-M195 or (111)In-M195, but the cytotoxic response among specimens was heterogeneous. CONCLUSION: NLS conjugation enhanced the nuclear uptake and cytotoxicity of (111)In-HuM195 and (111)In-M195 toward drug-resistant AML cell lines as well as patient specimens expressing a diversity of MDR phenotypes, including Pgp-170, BCRP1, or MRP1 transporters. Targeted Auger electron radioimmunotherapy using (111)In-labeled anti-CD33 monoclonal antibodies modified with NLS may be able to overcome MDR and provide a means of treating chemotherapy-resistant myeloid leukemias in patients.

Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery.

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy.

Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the nucleus. In this study, we have prepared a nuclear localization signal conjugated doxorubicin loaded Poly (D,L-lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and smooth surfaced nanoparticles. NPs (approximately 226 nm in diameter, 46% encapsulation efficiency) under in vitro conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity results showed that NLS conjugated NPs exhibited comparatively lower IC(50) value (2.3 microM/ml) than drug in solution (17.6 microM/ml) and unconjugated NPs (7.9 microM/ml) in breast cancer cell line MCF-7 as studied by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native DOX and unconjugated NPs. In conclusion, these results suggested that NLS conjugated doxorubicin loaded NPs could be potentially useful as novel drug delivery system for breast cancer therapy.

Recent improvements in the use of synthetic peptides for a selective photodynamic therapy.

Photodynamic therapy (PDT) is a relatively new cytotoxic treatment, predominantly used in anti-cancer approaches, that depends on the retention of photosensitizers in tumor and their activation after light exposure. Photosensitizers are photoactive compounds such as porphyrins and chlorins that upon photoactivation, effect strongly localized oxidative damage within the target cells. The ability to confine activation of the photosensitizer by restricting illumination to the tumor allows for a certain degree of selectivity. Nevertheless, the targeted delivery of photosensitizers to defined cells is a major problem in PDT of cancer, and one area of importance is photosensitizer targeting. Alterations or increased levels in receptor expression of specific cellular type occur in the diseased tissues. Therefore, photosensitizers can be covalently attached to molecules such as peptides, leading to a receptor-mediated targeting strategy. These active-targeting approaches may be particularly useful for anti-vascular PDT. Moreover, it has been shown that the photocytotoxicity of photodynamic drugs could be enhanced by delivering high amounts of a photosensitizer into subcellular organelles such as the nucleus where nucleic acids represent target molecules sensitive to photodamage. The recent progresses in the use of active-targeting strategy with synthetic peptides and the interest of using an active-targeting strategy in PDT, which could allow efficient cellular internalization of photosensitizers, are described in this review.

Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide.

We have recently described a new method for attaching padlock oligonucleotides to supercoiled plasmid DNA at specific sequences. A variant of this method has been developed in order to allow the coupling of targeting moieties to plasmids using a convenient strategy. After sequence-specific winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, the extremities of a triplex-forming oligonucleotide hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a hairpin oligonucleotide using T4 DNA ligase. Any targeting moiety may be attached to the hairpin oligonucleotide. This strategy was used to attach an NLS peptide to a luciferase-expressing plasmid. Despite the presence of the padlock oligonucleotide, the reporter gene was efficiently expressed after transfection of the plasmid in HeLa or T24 cells, using either cationic lipids or cationic polymers as transfecting agents. However, no increase in gene expression could be observed as a result of peptide attachment. Nevertheless, the coupling strategy described in this paper may find applications as a tool for plasmid functionalization in other targeting experiments, and may lead to the development of improved vectors for gene therapy.