Journal of
Materials Chemistry B
Trehalose-polyamine/DNA nanocomplexes:
impact of vector architecture on cell and organ
transfection selectivity†
A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and
trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been
synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to
enable systematic structural variations and, at the same time, ensuring that enantiomerically pure
vectors are obtained. Notably, changes in the molecular architecture translated into topological
differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of
regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments
further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results
highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve
targeting transfection without the need for any additional cell- or organ-sorting component.
click here to download all
BSV163/DOPE-mediated TRAIL gene transfection acts
synergistically with chemotherapy against cisplatinresistant ovarian cancer
Abstract
Ovarian cancer is the seventh most frequently diagnosed cancer among women
worldwide. Most patients experience recurrence and succumb eventually to resistant
disease, underscoring the need for an alternative treatment option. In
the presented manuscript, we investigated the effect of the TRAIL-gene,
transfected
by an innovative bioinspired lipid vector BSV163/DOPE in the presence
or absence of cisplatin, to fight against sensitive and resistant ovarian cancer.
We showed that BSV163/DOPE can transfect ovarian cancer cell lines (Caov3,
OVCAR3, and our new cisplatin-resistant,
CR-Caov3)
safely and efficiently. In
addition, TRAIL-gene
transfection in association with cisplatin inhibited cellular
growth more efficiently (nearly 50% in Caov3 cells after the combined treatment,
and 15% or 25% by each treatment alone, respectively) owing to an increase in
apoptosis rate, caspases activity and TRAIL’s death receptors expression. Most
importantly, such synergistic effect was also observed in CR-Caov3
cells demonstrated
by an apoptosis rate of 35% following the combined treatment in comparison
with 17% after TRAIL-gene
transfection or 6% after cisplatin exposition.
These results suggest this combination may have potential application for sensitive
as well as refractory ovarian cancer patients.
click here to download all
KEYWORDS
BSV163/DOPE, cisplatin-resistance,
lipid vector, ovarian cancer, TRAIL, transfection
An efficient low cost means
of biophysical gene transfection
in primary cells
Efficient, facile gene modification of cells has become an indispensable part of modern molecular
biology. For the majority of cell lines and several primary populations, such modifications can be
readily performed through a variety of methods. However, many primary cell lines such as stem
cells frequently suffer from poor transfection efficiency. Though several physical approaches have
been introduced to circumvent these issues, they often require expensive/specialized equipment
and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral
methods are capable of transducing difficult cellular populations, however such methods can be time
consuming for large arrays of gene targets, present biohazard concerns, and result in expression
of viral proteins; issues of concern for certain experimental approaches. We report here a widely
applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1–10 μl) cell
volumes and composed of equipment readily available to the average investigator. Using this system
we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to
commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process
improves the survival of sensitive stem cell populations while reducing reagent requirements for
protocols such as Cas9/gRNAs transfections.
click here to download all
Active transfection of genetic materials using
cyclodextrin-anchored nanovectors
Gene-based therapy is a sophisticated means for the treatment of various complex diseases like AIDS,
cancer, etc., as it resolves the genetic malfunction at the source instead of tackling the superficial
symptoms. However, the therapeutic, diagnostic, and theranostic potential of gene-based therapeutic
actives such as siRNA, mRNA, pDNA, aptamers, etc. is hindered by physicochemical as well as
physiological barriers in the form of insufficient bioavailability, systemic metabolism, rapid renal
clearance, inefficient carrier systems, etc. Although advanced carrier systems such as polyplexes,
lipoplexes, dendriplexes, hydrogels, polyrotaxanes, etc. are employed to overcome such challenges, their
structural configuration results in notable cytotoxicity to induce bio-incompatibility. In this context,
strategic integration of cyclodextrins subdues the cytotoxicity by virtue of unique architectural
characteristics and allows the fabrication of sophisticated systems for delivery of gene-based
therapeutics. Inclusion of cyclodextrins offers benefits like enhanced protection of gene-targeted
payloads, compact loading, nanoscale carrier dimensions, biostability, etc. by forming densely packed
cargo systems. Cyclodextrins nullify the active cationic moieties to lower in vivo cytotoxicity and
improve transfection efficiency across biomembranes. The multi-ligand binding capability of structurally-
modulated cyclodextrins avails receptor specificity and gene-targeted therapeutic efficiency. The ability
to form reversible covalent linkages allows the fashioning of multi-stimuli responsive supramolecular
nanocarriers for a desirable drug release profile. The present review article features cyclodextrins and
associated successful applications as the integral components of non-viral nanovectors such as cationic
polymers, dendrimers and polyrotaxanes as well as supramolecular assemblies for efficient delivery of
RNA-, DNA- and aptamer-based genetic payloads for the achievement of desired treatment outcomes.
click here to download all
In Vitro Photoselective Gene Transfection of Hepatocellular
Carcinoma Cells with Hypericin Lipopolyplexes
ABSTRACT: The lipopolyplex, a multicomponent nonviral gene
carrier, generally demonstrates superior colloidal stability, reduced
cytotoxicity, and high transfection efficiency. In this study, a new
concept, photochemical reaction-induced transfection, using photo-
sensitizer (PS)-loaded lipopolyplexes was applied, which led to
enhanced transfection and cytotoxic effects by photoexcitation of
the photosensitizer. Hypericin, a hydrophobic photosensitizer, was
encapsulated in the lipid bilayer of liposomes. The preformed
nanosized hypericin liposomes enclosed the linear polyethyleni-
mine (lPEI)/pDNA polyplexes, resulting in the formation of
hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP
containing 50 nM hypericin and 0.25 μg of pDNA were 185.6 ±
7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic
light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and
pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm2
was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate
dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed
photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity;
however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in
the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the
two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.
KEYWORDS: lipopolyplexes, photosensitizer, co-encapsulation, light-sensitive transfection, gene delivery
click here to download all
1shah-et-al-2024-in-vitro-photoselective-gene-transfection-of-hepatocellular-carc_219683
Selective Transfection of a Transferrin Receptor-Expressing Cell Line
with DNA−Lipid Nanoparticles
ABSTRACT: Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective
transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies.
Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity
continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex
interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these
additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic
expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that
eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster
(CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely
lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of
hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically
binds to hTfR1 and enables targeted transfection. The LNP’s pDNA encoded for the monomeric GreenLantern (mGL) reporter
protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal
particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering
to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry
based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a
5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were
mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVbneo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17−25% of
the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based
entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the
hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.
click here to download all
Electronic Journal of Biotechnology
abstract
Viral vectors are among the main approaches currently used in studies for executing gene delivery to target cells. During the past decades of active studies in gene therapy, including viruses, adenoviruses (Ads),
lentiviruses (LVs), and adeno-associated viruses (AAVs), have received the most attention among the biological approaches where potentially successful outcomes are recorded for numerous genetic conditions.
The success of delivery methods, however, remains unsatisfactory. Using some additives that can
improve transgene expression, transfection efficiency, viral particle production, and transduction efficiency is considered as a solution to overcoming the limitations of gene delivery approaches. These additives include caffeine, histone deacetylase (HDAC) inhibitors like sodium butyrate and valproic acid, and
polycationic agents like polybrene and protamine sulfate. In this review article, we present an overview
of viral vector-mediated retinal gene therapies and the application of some enhancers used to improve
the outcomes of gene delivery. Three routes of administrating viral vectors into ocular compartments
click here to download all