Salma N. Tammam

Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density

Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.

How successful is nuclear targeting by nanocarriers

The nucleus is ultimately the final target for many therapeutics treating various disorders including cancers, heart dysfunction and brain disorders. Owing to their specialized cell uptake and trafficking mechanisms, nanoparticles (NPs) allow drug targeting where degradation sensitive therapeutics could be delivered to their target tissues and cell in active form and sufficient concentration. However, it has recently become increasingly obvious that cytosolic internalization of a drug molecule does not entail its interaction with its subcellular target and hence careful nanoparticle design and optimization is required to enable nuclear targeting. This review, discusses the barriers to NP nuclear delivery; crossing the cell membrane, endo/lysosomal escape, cytoplasmic trafficking and finally nuclear entry focusing on how NP synthesis and modification could allow for bypassing each of the aforementioned barriers and successfully reaching the nucleus. Examples of nuclear targeted NPs are also discussed, stressing on the critical aspects of nuclear targeting and pointing out how the disease state might change the normal NP path and how such change could be exploited to increase efficiency of nuclear targeting. Finally, the criteria set for the evaluation of nanocarriers for nuclear delivery are discussed highlighting that quantitative rather than qualitative evaluation is required to evaluate how successful nanocarriers for nuclear delivery are, particularly with regards to the amount of drug delivered and released in the nucleus.

The effect of nanoparticle size and NLS density on nuclear targeting in cancer and normal cells; impaired nuclear import and aberrant nanoparticle intracellular trafficking in glioma

&NA; The cell nucleus is an interesting target in many diseases with particular interest in cancer. Previously, nuclear targeted small and large chitosan nanoparticles (S‐NPs ≈ 25 nm, and L‐NPs ≈ 150 nm respectively), modified with low, intermediate and high densities of NLS (L‐NLS, I‐NLS and H‐NLS) were developed and assessed in L929 fibroblasts. However, to evade apoptosis and stimulate tumor growth cancer cells are capable of manipulating the nuclear‐cytoplasmic transport on many levels, making NPs that are capable of nuclear targeting in normal cells incapable of doing so in cancer. For such reason, here, the nuclear delivery efficiency of S‐NPs and L‐NPs was assessed as a function of their NLS density in cancer and non‐cancer cells. For S‐NPs, in all cells tested, NLS was unnecessary for nuclear delivery; unmodified S‐NPs showed higher nuclear delivery than NLS‐S‐NPs due to their ability to gain nuclear entry in a passive manner. For L‐NPs, L‐NLS‐L‐NPs showed ≈ 8.5, 33, 1.8 and 7.2 fold higher nuclear deliveries than H‐NLS‐L‐NPs in L929 fibroblasts, primary human fibroblasts, HEK 293 and lung cancer cells, respectively. In glioma however, unmodified L‐NPs showed highest nuclear delivery, whereas NLS‐L‐NPs were retained in the cytoplasm. Experiments conducted in the presence of inhibitors of the classical nuclear import pathway indicated that due to overexpression of importin &agr;, classical nuclear import in glioma is impaired leading to aberrant NP intracellular trafficking and nuclear import. Graphical abstract Figure. No caption available.

Development of an inhalable, stimuli-responsive particulate system for delivery to deep lung tissue.

Lung cancer, the deadliest solid tumor among all types of cancer, remains difficult to treat. This is a result of unavoidable exposure to carcinogens, poor diagnosis, the lack of targeted drug delivery platforms and limitations associated with delivery of drug to deep lung tissues. Development of a non-invasive, patient-convenient formula for the targeted delivery of chemotherapeutics to cancer in deep lung tissue is the aim of this study. The formulation consisted of inhalable polyvinylpyrrolidone (PVP)/maltodextrin (MD)-based microparticles (MPs) encapsulating chitosan (CS) nanoparticles (NPs) loaded with either drug only or drug and magnetic nanoparticles (MNPs). Drug release from CS NPs was enhanced with the aid of MNPs by a factor of 1.7 in response to external magnetic field. Preferential toxicity by CS NPs was shown towards tumor cells (A549) in comparison to cultured fibroblasts (L929). The prepared spray freeze dried (SFD) powders for CS NPs and CS MNPs were of the same size at ∼6μm. They had a fine particle fraction (FPF≤5.2μm) of 40-42% w/w and mass median aerodynamic diameter (MMAD) of 5-6μm as determined by the Next Generation Impactor (NGI). SFD-MPs of CS MNPs possess higher MMAD due to the high density associated with encapsulated MNPs. The developed formulation demonstrates several capabilities including tissue targeting, controlled drug release, and the possible imaging and diagnostic values (due to its MNPs content) and therefore represents an improved therapeutic platform for drug delivery to cancer in deep lung tissue.

A high throughput method for quantification of cell surface bound and internalized chitosan nanoparticles.

Chitosan has become a popular polymer for drug delivery. Its hydro solubility and mild formulation conditions have made it an attractive polymer for macromolecular delivery. Accurate quantification of internalized chitosan nanoparticles (NPs) is imperative for fair assessment of the nano-formulation where it is important to determine the exact amount of drug actually being delivered into the cell, especially for macromolecular drugs where cellular entry is limited by molecule size and/or charge. The preferential affinity of wheat germ agglutinin tagged with fluorescein isothiocyanate (WGA-FITC) to chitosan is exploited in the development of a simple and rapid method for the differentiation between and quantification of cell surface bound and internalized chitosan NPs. The percentage of cell surface bound NPs could be easily determined and corrected NP uptake could be calculated accordingly. The developed method is applicable in several cell lines and has successfully been tested with NPs with different sizes (25 and 150nm) and with very low NP concentrations (20μg/mL). The method will allow for the correct evaluation of chitosan NP uptake and could be further used to evaluate chitosan based nanomedicine and provide guidelines on how to modify NPs for enhanced internalization, and improved drug delivery.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

A single tube system for the detection of Mycobacterium tuberculosis DNA using gold nanoparticles based FRET assay

The global combat against MTB is limited by challenges in accurate affordable detection. In this study, a rapid, affordable, single tube system for detection of unamplified MTB16s rDNA was developed. Utilizing a AuNP based FRET system, this assay achieved a sensitivity and specificity of 98.6% and 90% respectively.

Chitosan gold nanoparticles for detection of amplified nucleic acids isolated from sputum

A platform for nucleic acid detection employing chitosan and chitosan coated gold nanoparticles (AuNPs) was developed. Mycobacterium tuberculosis (MTB) was used as a model target. MTB DNA was extracted from sputum using simple total nucleic acid extraction. Following amplification of MTB DNA, chitosan and AuNPs were added to samples. Free chitosan conjugated non-target DNA in negative samples, avoiding AuNP-DNA interaction and hence negative samples remained red. In positive samples, amplified DNA was capable of saturating free chitosan leading to AuNP aggregation where positive samples turned blue. Via visual color detection 15/16 MTB positive samples and 3/3 negative samples were correctly identified. This test is a 1-tube, 1-step assay reducing the risk of contamination in molecular laboratories and is a proof of concept on how chitosan; a cheap polymer could increase the sensitivity of AuNPs towards specific detection of nucleic acids without using target specific oligotargeters or expensive extraction kits.

Nuclear and cytoplasmic delivery of lactoferrin in glioma using chitosan nanoparticles: Cellular location dependent-action of lactoferrin

Graphical abstract Figure. No caption available. &NA; Lactoferrin (Lf) exerts anti‐cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti‐cancer effect. Upon treatment with 300 and 800 &mgr;g/mL Lf loaded chitosan NPs, nuclear targeted Lf‐NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf‐NPs at 300 &mgr;g/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti‐cancer protein.