15 research outputs found
Insight into the Role of Physicochemical Parameters in a Novel Series of Amphipathic Peptides for Efficient DNA Delivery
Amphipathic peptides constitute a
class of molecules with the potential
to develop as efficient and safer alternatives to viral and other
nonviral vectors for intracellular delivery of therapeutics. These
peptides can be useful for nucleic acid delivery and hence promise
to have pharmaceutical application, particularly in gene therapy.
In order to design novel amphipathic peptides and improve their efficiency
of therapeutic cargo delivery, one needs to understand the role of
the physicochemical properties of the peptide. There are very few
reports in the literature where the physicochemical properties of
the peptide have been correlated with efficiency of plasmid DNA delivery.
In the present work we hunted out a naturally occurring amphipathic
peptide termed Mgpe-1 (derived from HUMAN Protein phosphatase 1E)
as a possible novel DNA delivery agent. We systematically altered
the physicochemical parameters of this peptide to further enhance
its DNA delivery efficiency. We changed its amphipathicity (from secondary
to primary), the total charge (from +6 to +9), hydrophobicity, and
the amino acid composition (lysine and serines to arginine; substitution
of tryptophan) and studied which of these alterations affect DNA delivery
efficiency. Our results showed that although Mgpe-1 exhibited very
strong cellular uptake, its plasmid DNA delivery efficiency was poor.
The presence of nine arginines improved the DNA delivery efficiency,
and the effect was observed in both the primary and the secondary
amphipathic variants. We further observed that the presence of tryptophan
was important but not essential and the effect of its removal was
stronger in the case of the secondary amphipathic peptide. However,
increase in total hydrophobicity of the peptide led to a fall in transfection
efficiency in the primary amphipathic peptide whereas the secondary
amphipathic peptide having the same chemical composition was almost
unaffected by this change. The primary amphipathic peptides with high
positive charge and low hydrophobicity formed colloidally stable polyplexes
with DNA and avoided a major impediment in DNA delivery, namely, the
aggregation of polyplexes and cytotoxicity. The secondary amphipathic
variants by virtue of the positional arrangement of the amino acids
led to formation of polyplexes with partly hydrophilic surfaces which
prevented aggregation and controlled particle size irrespective of
the hydrophobicity. Two variants in the series Mgpe-3 and Mgpe-4 having
nine positive charges with less hydrophobicity showed high transfection
efficiency in multiple cell lines along with serum stability and much
less cytotoxicity and promise to be novel and efficient DNA delivery
vectors
Zinc Oxide Nanoparticles Dispersed in Ionic Liquids Show High Antimicrobial Efficacy to Skin-Specific Bacteria
Zinc oxide (ZnO) nanoparticles have
been shown
in the literature to have antibacterial properties and have been widely
used in antibacterial formulations. However, one of the problems with
ZnO nanoparticles is their tendency to aggregate, thereby causing
damage to normal cells and lowering their antibacterial efficacy during
application. In this work, we have attempted to avoid this by using
a combination of ZnO nanoparticles and ionic liquids, a class of low
melting salts containing organic cations and organic/inorganic anions
that show antibacterial property as well, and tested the antibacterial
activity of this dispersion. ZnO nanoparticles of 60 nm were dispersed
in two different ionic liquidscholine acetate (IL1) and 1-butyl-3-methylimidazolium
chloride (IL2)to achieve high dispersibility, whereas ZnO
dispersed in phosphate-buffered saline was taken as a control. These
dispersions were tested on four strainsEscherichia
coli, Bacillus subtilis, Klebsiella pneumoniae, and Staphylococcus epidermidis. Maximum efficiency was
obtained for ZnO nanoparticles dispersed in imidazolium-based ionic
liquids against skin-specific S. epidermidis. Skin infections induced by S. epidermidis are prevalent in hospital-acquired diseases. In most cases, traditional
antibiotic-based therapies fail to combat such infections. Our strategy
of developing a dispersion of ZnO nanoparticles in ionic liquids shows
superior antibacterial efficacy in comparison to that shown individually
by ZnO nanoparticles or ionic liquids. We have also established that
the mechanism of killing this skin-specific bacterium is possibly
through the production of reactive oxygen species leading to bacterial
cell lysis. Further, we showed that this formulation is biocompatible
and nontoxic to normal keratinocyte cells even under coculture conditions
Deciphering the Role of Chondroitin Sulfate in Increasing the Transfection Efficiency of Amphipathic Peptide-Based Nanocomplexes
Glycosaminoglycans, both cell-surface
and exogenous, can interfere
with DNA delivery efficiency of nonviral carrier systems. In this
work, we report an extensive comparative study to explore the effect
of exogenously added chondroitin sulfate on biophysical characteristics,
cellular uptake, transfection efficiency, and intracellular trafficking
of nanocomplexes formed using primary and secondary amphipathic peptides
developed in our laboratory. Our results indicate that the presence
of exogenous chondroitin sulfate exhibits differential enhancement
in transfection efficiency of the amphipathic peptides depending upon
their chemical nature. The enhancement was more pronounced in primary
amphipathic peptide-based nanocomplexes as compared to the secondary
counterpart. This difference can be attributed to possible alteration
of the intracellular entry pathway in addition to increased extracellular
stability, less cellular toxicity, and assistance in nuclear accumulation.
These results imply potential use of glycosaminoglycans such as chondroitin
sulfate to improve the transfection efficiency of primary amphipathic
peptides for possible in vivo applications
Albumin Nanoparticles Surface Decorated with a Tumor-Homing Peptide Help in Selective Killing of Triple-Negative Breast Cancer Cells
In this article, we describe a method of delivery of
doxorubicin
using a novel tumor-homing peptide-based albumin nanoparticle system
to triple-negative breast cancer cells (TNBC). The absence and reduced
expression of the hormone (estrogen, progesterone) and HER2 (human
epidermal growth factor 2) receptors, respectively, render TNBC patients
nonsusceptible to different available targeted therapies. These peptide-modified
nanoparticles could be taken up by TNBC cells more effectively than
their bare counterparts. The drug-loaded peptide-modified nanoparticles
achieved an optimal but crucial balance between cell killing in cancerous
cells and cell survival in the noncancerous ones. This appears to
be because of different routes of entry and subsequent fate of the
bare and peptide-modified nanoparticles in cancerous and noncancerous
cells. In a TNBC mouse model, the peptide-modified system fared better
than the free drug in mounting an antitumor response while not being
toxic systemically
Integration Host Factor of <i>Mycobacterium tuberculosis</i>, mIHF, Compacts DNA by a Bending Mechanism
<div><p>The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of <i>E. coli</i> and mycobacterial IHF in DNA compaction.</p></div
CD profile of mIHF-80.
<p>A CD analysis of mIHF-80 is suggestive of a globular, folded protein. The protein appears to be primarily alpha-helical and the helical content calculated to be more than 85% of the total secondary structure content of the protein.</p
Representative AFM images of protein-DNA complexes formed between mIHF-80 and negatively supercoiled plasmid DNA.
<p>(A) Plasmid DNA in the absence of mIHF-80. (B)–(I): mIHF-80-pPROEX-HTc complexes with increasing mIHF-80 dimers per 10 base pair of DNA as follows; (B) 1, (C) 2, (D&E) 4, (F & G) 8. The scale of all images in A to G is (3 µm×3 µm). (H) & (I): Rigid nucleoprotein filaments identified in (F) and (G) above are shown at a slightly magnified scale of (1 µm×1 µm). The protein complexes are marked by arrows.</p
EMSA for nucleic acid binding.
<p>Binding of mIHF-80 to (a) circular and (b) linear DNA was analyzed by running the reaction mixture on agarose gel and visualizing by EtBr staining. mIHF-80 binds DNA non-specifically in a concentration-dependent manner. (a) In the reaction mix, 18, 36, 75, 150, 300, 450, 600 or 900, (lanes 2–9) were incubated with circular supercoiled DNA at 37°C for 60 min. (b) To monitor binding to linear DNA, a similar reaction was carried out with 18, 37, 75, 150, 300, 450, 600 or 900 ng mIHF-80 (lanes 2–9) to monitor gel shift. Lane 1 is control with no protein in both gels.</p
Representative AFM images of protein-DNA complexes formed between mIHF-80 and linear DNA.
<p>(A) Linear pPROEX-HTc in the absence of mIHF-80. (B)–(D): mIHF-80-pPROEX-HTc (linearized) complexes with following mIHF-80 dimer molecules per 10 base pairs of DNA; (B) 2, (C) 4 and (D) 8. The scale of all images is (3 µm×3 µm).</p
Geometrical parameters of linear DNA+ mIHF-80 complexes.
a<p>Indicated values are averages of molecules analyzed (N).</p>b<p>σ is the Standard deviation for each parameter calculated over molecules analyzed (N).</p